bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide sequence of the mRNA encoding the pre-alpha-subunit of mouse thyrotropin.
Authors: Chin W W , Kronenberg H M , Dee P C , Maloof F , Habener J F
Published: May 2006

We have constructed and cloned in bacteria recombinant DNA molecules containing DNA sequences coding for the precursor of the alpha subunit of thyrotropin (pre-TSH-alpha). Double-stranded DNA complementary to total poly(A)+RNA derived from a mouse pituitary thyrotropic tumor was prepared enzymatically, inserted into the Pst I site of the plasmid pBR322 by using poly(dC).poly(dG) homopolymeric extensions, and cloned in Escherichia coli chi 1776. Cloned cDNAs encoding pre-TSH-alpha were identified by their hybridization to pre-TSH-alpha mRNA as determined by cell-free translations of hybrid-selected and hybrid-arrested RNA. The nucleotide sequences of two cDNAs (510 and 480 base pairs) were determined with chemical methods and corresponded to much of the region coding for the alpha subunit and the 3' untranslated region of pre-TSH-alpha mRNA. The sequence of the 5' end of the mRNA was determined from cDNA synthesized by using total mRNA as template and a restriction enzyme DNA fragment as primer. Together these sequences represented greater than 90% of the coding and noncoding regions of full-length pre-TSH-alpha mRNA, which was determined to be 800 bases long. The amino acid sequence of the pre-TSH-alpha deduced from the nucleotide sequence showed a NH2-terminal leader sequence of 24 amino acids followed by the 96-amino-acid sequence of the apoprotein of TSH-alpha. There is greater than 90% homology in the amino acid sequences among the murine, ruminant, and porcine alpha subunits and 75-80% homology among the murine, equine, and human alpha subunits. Several regions of the sequence remain absolutely conserved among all species, suggesting that these particular regions are essential for the biological function of the subunit. The successful cloning of the alpha subunit of TSH will permit further studies of the organization of the genes coding for the glycoprotein hormone subunits and the regulation of their expression.
Increased levels of threonyl-tRNA synthetase in a borrelidin-resistant Chinese hamster ovary cell line.
Authors: Gantt J S , Bennett C A , Arfin S M
Published: May 2006

The growth of Chinese hamster ovary cells in medium containing reduced concentrations of threonine is inhibited by borrelidin, a macrolide antibiotic. Borrelidin-resistant clones have been isolated after ethyl methanesulfonate mutagenesis. One clone, 1C-1, has a 3-fold increased level of threonyl-tRNA synthetase [L-threonine:tRNAThr ligase (AMP-forming), EC 6.1.1.3] as determined by both activity measurements and antiserum titrations. The levels of four other aminoacyl-tRNA synthetases and of tRNAThr are the same in strain 1C-1 and in the wild-type parent. The phenotype of increased threonyl-tRNA synthetase activity is recessive to wild type in cell hybrids.
Binding of ligands to the active site of carboxypeptidase A.
Authors: Rees D C , Lipscomb W N
Published: May 2006

We compare the detailed binding modes of the 39-amino acid inhibitor from potatoes, glycyl-L-tyrosine, the ester analogue CH3OC6H4(CO)CH2CH(CO2(-))C6H5, and indole acetate to the exopeptidase carboxypeptidase A (EC 3.4.17.1). In the potato inhibitor, cleavage of the COOH-terminal glycine-39 leaves a new carboxylate anion of valine-38 having one oxygen on zinc and the other as a receptor of a hydrogen bond from tyrosine-248 of carboxypeptidase. Tyrosine-248 also receives a hydrogen bond from the amide proton of the originally penultimate peptide bond between tyrosine-37 and valine-38. This hydrogen bond suggests product stabilization which is available to peptides and depsipeptides but not to esters lacking an equivalent peptide bond (nonspecific esters). Also, this structure may represent the intermediate binding step for the uncleaved substrate as it moves along the binding subsites. In particular, this may be the binding mode for the substrate after association of the COOH-terminal region of the substrate with the residues at binding subsite S2 (tyrosine-198, phenylalanine-279, and arginine-71) and preceding entry into the catalytic site S1'. These stabilized complexes allow some understanding of the effect of indole acetate, shown here to bind in the pocket at S1', as a competitive inhibitor for esters (for which entry into S1' precedes the rate-determining catalytic step for hydrolysis) and as a noncompetitive inhibitor for peptides (for which entry into S1' is rate limiting). These results, including the binding mode of the ester analogue, are consistent with the original proposal from x-ray studies that both esters and peptides are cleaved with the carboxy terminus at S1', although not necessarily by the same chemical steps.
Estrogen formation in stromal cells of adipose tissue of women: induction by glucocorticosteroids.
Authors: Simpson E R , Ackerman G E , Smith M E , Mendelson C R
Published: May 2006

Stromal cells prepared from adipose tissue of women were maintained in monolayer culture to study the regulation of aromatase activity by hormones. Aromatase activity was stimulated 20- to 100-fold by dexamethasone at a concentration of 250 nM. Half-maximal stimulation of aromatase activity was attained at a dexamethasone concentration of 2.7 nM. The stimulatory effect of dexamethasone was apparent after a preincubation time of 4 hr, and stimulation was maximal after 24 hr of preincubation. The stimulatory effect of dexamethasone was observed only when fetal calf serum also was present in the culture medium. Of the various steroids tested, dexamethasone was the most potent in stimulating aromatase activity. Cortisol was less effective than dexamethasone, whereas corticosterone, at a concentration of 250 nM, caused only a small stimulation of aromatase activity. Progesterone and deoxycorticosterone (250 nM) did not affect aromatase activity. Cytosolic fractions prepared from stromal cells that had been maintained in monolayer culture were found to contain a homogenous population of sites that specifically bound [3H]dexamethasone with relatively high affinity (Kd = 2.9 nM) and low capacity (38 fmol per mg of protein). The stimulatory effect of dexamethasone on aromatase activity was prevented by simultaneous incubation with cortisol 21-mesylate (0.1-10 microM), a compound known to block the binding of glucocorticosteroids to cytoplasmic receptors. The stimulatory effect of dexamethasone also was prevented by incubation of the cells with cycloheximide or actinomycin D. These findings are suggestive that glucocorticosteroids act to increase aromatase activity in stromal cells by inducing the synthesis of new enzyme protein.
Interactions of low density lipoprotein receptors with coated pits on human fibroblasts: estimate of the forward rate constant and comparison with the diffusion limit.
Authors: Goldstein B , Wofsy C , Bell G
Published: May 2006

We present a theoretical study of the interaction of low density lipoprotein (LDL) receptors with coated pits. From published experiments we estimate that the forward rate constant, k+, for the binding of a LDL receptor to a coated pit on a human fibroblast at 37 degrees C is greater than or equal to 3 X 10(-10) cm2/sec, and the mean time an LDL receptor spends on the cell surface before being captured by a coated pit, tc, is less than or equal to 1.8 min. If, when an LDL receptor enters, it remains within the coated pit until the coated pit pinches off to form a coated vesicle, then k+ = 3 X 10(-10) cm2/sec and tc = 1.8 min. We derive expressions for the diffusion limit of k+ and tc when particles (LDL receptors) diffuse in two dimensions until they hit and are absorbed by circular absorbers (coated pits) that have finite lifetimes. The absorbers appear and disappear at equal rates so that their concentration remains constant. We use these expressions to show that a diffusion limit of k+ = 3 X 10(-10) cm2/ sec would be obtained if D, the diffusion coefficient for an LDL receptor on a human fibroblast at 37 degrees C equaled 3.3 X 10(-11) cm2/sec. Because this value is in agreement with the experimentally determined value for D, we conclude that random insertion of LDL receptors into the plasma membrane, followed by pure diffusional motion of LDL receptors on the cell surface until they are irreversibly absorbed in coated pits, is consistent with experiment.
Rates of change in quantitative traits from fixation of new mutations.
Authors: Hill W G
Published: May 2006

Expressions are derived for the response to directional selection for a quantitative trait that comes from fixation of new mutations in a finite population. For additive genes with a distribution of mutant gene effects symmetric about zero, the response from fixing mutations occurring in a single generation is 2 Ni sigma 2M/sigma, in which N is the effective population size, i is the selection intensity, sigma is the phenotypic standard deviation, and sigma 2M is the increment in variance in the generation immediately after occurrence of the mutations. This response is 2N times that immediately after occurrence of the mutations. With continuous mutation each generation, the asymptotic rate of response is also 2Ni sigma 2M/sigma and the asymptotic variance is independent of i. For completely dominant mutations with symmetric effects, the rates are Ni sigma 2M/sigma; and for recessive mutations the rates are proportional to (Ni)1/2. If the distribution of mutant gene effects, a, is not symmetric about zero, responses depend on the mean square of effects of mutations with positive effect, rather than on the variance of their effects. Rates of change in fitness and of traits correlated with fitness are also analyzed. It is argued that new mutations have contributed substantially to long-term responses in many laboratory selection experiments.
Stimulated macrophages express a new glycoprotein receptor reactive with Griffonia simplicifolia I-B4 isolectin.
Authors: Maddox D E , Shibata S , Goldstein I J
Published: May 2006

This paper presents data on reactions of murine macrophages with a variety of lectins, with special focus on Griffonia simplicifolia I-B4 isolectin, the only lectin we tried that distinguishes stimulated macrophages from resident populations. Specificity of Griffonia simplicifolia I reaction with carbohydrate determinants at the cell surface is shown by (i) ability of alpha-galactosidase treatment of intact cells to abolish all lectin binding whereas beta-galactosidase has no effect on lectin binding, (ii) ability of methyl alpha-D-galactopyranoside to completely inhibit lectin binding with methyl alpha-D-galactopyranoside having no effect on lectin binding, (iii) ability of brief treatment of intact cells with trypsin to liberate a glycopeptide but reacts with G. simplicifolia I to form a precipitate that is dissolved by addition of methyl-alpha-D-galactopyranoside or alpha-galactosidase, (iv) ability of methyl alpha-D-galactopyranoside (but no other monosaccharide) to completely inhibit avid binding of macrophages to G. simplicifolia I lectin immobilized on an insoluble support, and (v) ability of immobilized lectin to separate macrophages into highly pure subpopulations of lectin-reactive and lectin-unreactive cells, as shown by examination of fluorescein-labeled lectin-treated cells with phase-contrast/fluorescence microscopy.
Interferon increases HLA synthesis in melanoma cells: interferon-resistant and -sensitive cell lines.
Authors: Basham T Y , Bourgeade M F , Creasey A A , Merigan T C
Published: May 2006

We report that human leukocyte interferon preparations increase the expression of beta 2-microglobulin by 100-200% on the surface of normal fibroblast and melanoma cell lines sensitive to interferon. This increase in expression can be correlated with an increase in HLA synthesis as measured by incorporation of [35S]methionine in these antigens. This enhanced HLA synthesis, which is 5- to 17-fold, is time dependent and dose related. Synchronized cells in the G0/G1 phase of the cell cycle appear to be more sensitive to this interferon action. Neither an increase in surface expression nor in HLA synthesis is observed in a melanoma cell line resistant to the antiviral and antigrowth effects of interferon. Furthermore, there appears to be a stronger correlation between this increased HLA synthesis and the antiviral function than between it and the antiproliferative action of interferon.
Thiamin-responsive maple-syrup-urine disease: decreased affinity of the mutant branched-chain alpha-keto acid dehydrogenase for alpha-ketoisovalerate and thiamin pyrophosphate.
Authors: Chuang D T , Ku L S , Cox R P
Published: May 2006

The biochemical basis for the therapeutic effects of thiamin in thiamin-responsive maple-syrup-urine disease (MSUD) was investigated in intact and disrupted fibroblast cultures from normals and patients with various forms of MSUD. Decarboxylation of alpha-keto[1-14C]isovalerate (KIV) by intact cells from a thiamin-responsive MSUD patient was at 30-40% of the normal rate with or without thiamin in the incubation medium. Under similar conditions, intact classical MSUD fibroblasts failed to decarboxylate KIV. Branched-chain alpha-keto acid (BCKA) dehydrogenase activity measured in disrupted cells from the thiamin-responsive subject showed sigmoidal kinetics in the absence of thiamin pyrophosphate (TPP), with an increased concentration of substrate needed for half-maximal velocity (K0.5 for KIV = 7 mM vs. 0.05 mM in normal cells). When assayed with 0.2 mM TPP present, the mutant enzyme showed (i) a shift in kinetics to near Michaelis-Menten type as observed with the normal BCKA dehydrogenase and (ii) a lower K0.5 value of 4 mM for KIV, suggesting a TPP-mediated increase in the mutant enzyme's affinity for substrate. By contrast, TPP increased only the Vmax and was without effect on the apparent Km for KIV of the BCKA dehydrogenase from cells of normals and patients with classical MSUD and variant thiamin-responsive MSUD (grade 3). Measurement of the apparent Km for TPP of the BCKA dehydrogenase from thiamin-responsive mutant MSUd cells showed a 16-fold increase in the constant to 25 microM compared to enzymes from normal or classical MSUD cells. These findings demonstrate that the primary defect in the thiamin-responsive MSUD patient is a reduced affinity of the mutant BCKA dehydrogenase for TPP that results in impaired oxidative decarboxylation of BCKA.
Inactivation of cap-binding proteins accompanies the shut-off of host protein synthesis by poliovirus.
Authors: Lee K A , Sonenberg N
Published: May 2006

Infection of HeLa cells with poliovirus results in a rapid shut-off of host protein synthesis. It has been suggested that inactivation of a protein that binds to the cap structure of cellular mRNAs would explain the selective inhibition of host protein synthesis because the naturally uncapped poliovirus RNA can be translated by a cap-independent mechanism. To test directly for the presence of cap-binding proteins in poliovirus-infected and mock-infected cells, we analyzed initiation factor preparations for their ability to specifically crosslink to the 5' cap structure of oxidized reovirus mRNA. The data presented here show that the crosslinking ability of the different cap-binding proteins (24-, 28-, 32-, 50-, and 80-kilodalton polypeptides) is reduced in preparations from poliovirus-infected as compared to mock-infected cells. This reduction correlates with the inability of initiation factor preparations from infected cells to restore translation of capped mRNAs in extracts of poliovirus-infected cells. In addition, initiation factor preparations from poliovirus-infected cells have the ability to rapidly inactivate cap-binding proteins and can also impair the restoring activity of initiation factors from mock-infected cells.
NMR chemical shift imaging in three dimensions.
Authors: Brown T R , Kincaid B M , Ugurbil K
Published: May 2006

A method for obtaining the three-dimensional distribution of chemical shifts in a spatially inhomogeneous sample using Fourier transform NMR is presented. The method uses a sequence of pulsed field gradients to measure the Fourier transform of the desired distribution on a rectangular grid in (k,t) space. Simple Fourier inversion then recovers the original distribution. An estimated signal/noise ratio of 20 in 10 min is obtained for an "image" of the distribution of a 10 mM phosphorylated metabolite in the human head at a field of 20 kG with 2-cm resolution.
Electrostatic potential molecular surfaces.
Authors: Weiner P K , Langridge R , Blaney J M , Schaefer R , Kollman P A
Published: May 2006

Color-coded computer graphics representations of the electrostatic potentials of trypsin, trypsin-inhibitor, prealbumin and its thyroxine complex, fragments of double-helical DNA, and a netropsin--DNA complex illustrate the electrostatic and topographic complementarity in macromolecule-ligand interactions. This approach is powerful in revealing intermolecular specificity and shows promise of having predictive value in drug design.
A scanning calorimetric study of small molecule-lipid bilayer mixtures.
Authors: Sturtevant J M
Published: May 2006

The influence of small molecules on the main phase transition of dimyristoyl phosphatidylcholine was observed by high-sensitivity differential scanning calorimetry. The variation with temperature of the excess heat capacity is markedly affected by solid solution formation, which appears to be of frequent occurrence in solutions in lipid bilayers. The experimental results are in some cases in reasonable agreement with ideal solution theory.
Proteolytic processing of poliovirus polypeptides: antibodies to polypeptide P3-7c inhibit cleavage at glutamine-glycine pairs.
Authors: Hanecak R , Semler B L , Anderson C W , Wimmer E
Published: May 2006

Proteolytic processing of poliovirus polypeptides was examined by the addition of antibodies directed against the viral proteins P3-7c and P2-X to a cell-free translation extract prepared from infected HeLa cells. Antisera to P3-7c specifically inhibited in vitro processing at Gln-Gly pairs. Partial amino acid sequence analysis revealed a second Tyr-Gly pair that is utilized in protein processing. Neither Tyr-Gly cleavage is affected by antibody to P3-7c. Anti-P3-7c antibodies react not only with P3-7c but also with P3-6a and P3-2, two viral polypeptides NH2-coterminal with P3-7c. Preimmune and anti-P2-X antibodies had no effect on the processing of poliovirus proteins in vitro. We conclude that the activity responsible for processing poliovirus polypeptides at Gln-Gly pairs resides in the primary structure of P3-7c (or P3-2 and P3-6a) and not in P2-X.
Dynamical theory of activated processes in globular proteins.
Authors: Northrup S H , Pear M R , Lee C Y , McCammon J A , Karplus M
Published: May 2006

A method is described for calculating the reaction rate in globular proteins of activated processes such as ligand binding or enzymatic catalysis. The method is based on the determination of the probability that the system is in the transition state and of the magnitude of the reactive flux for transition-state systems. An "umbrella sampling" simulation procedure is outlined for evaluating the transition-state probability. The reactive flux is obtained from an approach described previously for calculating the dynamics of transition-state trajectories. An application to the rotational isomerization of an aromatic ring in the bovine pancreatic trypsin inhibitor is presented. The results demonstrate the feasibility of calculating rate constants for reactions in proteins and point to the importance of solvent effects for reactions that occur near the protein surface.
Mass mapping of a protein complex with the scanning transmission electron microscope.
Authors: Engel A , Baumeister W , Saxton W O
Published: May 2006

A mass map of the hexagonally packed intermediate layer (HPI-layer), a regular protein monolayer from the cell envelope of Micrococcus radiodurans, has been obtained by scanning transmission electron microscopy. Samples were freeze-dried within the microscope, and low-dose images were recorded in the dark-field mode directly in digital form and processed by correlation averaging. The averaged projection of the unstained structure--i.e., the mass map--thus calculated shows a resolution to 3-nm period and reveals morphological features consistent with those obtained by negative staining. The mass of individual morphological domains was extracted by using variously the mass map itself or an average from a negatively stained HPI layer to define the domain boundaries. Protrusions as small as 1,300 daltons could be measured reproducibly within the unit cell of 655,000 daltons. The method developed opens an avenue to identify molecular species in situ and to correlate topographic information with biochemical data.
Interferon enhancement of the invasive capacity of Ewing sarcoma cells
in vitro
Authors: Siegal Gene P. , Thorgeirsson Unnur P. , Russo Raimondo G. , Wallace Donald M. , Liotta Lance A. , Berger Shelby L.
Published: May 2006

The ability of interferons to reduce cell proliferation
in vitro
and
in vivo
is a well-studied phenomenon. To extend such observations, the effect of interferons on the invasiveness
in vitro
of human malignant cells derived from a Ewing sarcoma was evaluated. Two related parameters were examined: (
i
) production of type IV (basement membrane) collagenase and (
ii
) penetration of human amnion basement membrane and collagenous stroma. After 6 days of treatment with crude fibroblast, leukocyte, or lymphoblastoid interferon at 100 units/ml in serum-free medium, type IV collagenase levels increased 2- to 4-fold per cell relative to those of untreated controls. With homogeneous fibroblast and lymphoblastoid interferons, a 2-fold elevation in type IV collagenase was detected after 2 days, with further increases, occasionally dramatic, occurring on the 4th and 6th day of treatment. The ability of Ewing sarcoma cells to invade human amnion connective tissue was measured after 6 days of treatment with various interferons. Relative to the behavior of untreated controls, crude leukocyte interferon, homogeneous lymphoblastoid interferon, and homogeneous fibroblast interferon at 100 units/ml augmented invasiveness 3-, 17- and 22-fold, respectively, when cells were allowed 4 days in which to traverse the amnion. When untreated cells were exposed simultaneously to the amnion and to homogeneous lymphoblastoid or fibroblast interferon, a 4- to 5-fold increase in invasiveness above control levels was observed in 2 days. These data emphasize the complexity of interferon-induced phenomena. In any overview, the effects of interferon on both the tumor cell and the host must be considered.
Fibronectin binds to the C1q component of complement.
Authors: Bing D H , Almeda S , Isliker H , Lahav J , Hynes R O
Published: May 2006

Fibronectin immobilized to plastic tubes binds soluble C1q with a Kd of 82 +/- 2.6 nM. The binding of fibronectin to C1q is relatively insensitive to pH but is sensitive to ionic conditions. C1q covalently bound to Sepharose selectively binds cellular fibronectin produced by a hamster fibroblast cell line. The globular head regions of C1q have no effect on the binding of C1q to fibronectin but the collagenous tails of C1q interfere competitively with a Ki of 59 nM. We conclude that fibronectin binds C1q via its collagen-like tail region and thus the process resembles the binding of fibronectin to gelatin. This is further emphasized by our observation that gelatin binds to fibronectin immobilized on plastic tubes with a Kd of 131 nM. Because fibronectin stimulates endocytosis in several systems and promotes the clearance of particulate material from the circulation, these results suggest the possibility that fibronectin could function in the clearance of C1q-coated material such as immune complexes or cellular debris.
Reduced availability of endogenously synthesized methionine for S-adenosylmethionine formation in methionine-dependent cancer cells.
Authors: Coalson D W , Mecham J O , Stern P H , Hoffman R M
Published: May 2006

Methionine (Met) dependence--i.e., the inability of cultured cells to grow when Met is replaced by its immediate precursor homocysteine (Met-Hcy+ medium)--is a frequent component of the oncogenically transformed phenotype. Normal cells, on the other hand, grow in this medium. There have been reports [Hoffman, R. M. & Erbe, R. W. (1976) Proc. Natl. Acad. Sci. USA 73, 1523-1527; Hoffman, R. M., Jacobsen, S. J. & Erbe, R. W. (1978) Biochem. Biophys. Res. Commun. 82, 228-234] of normal or higher rats of Met biosynthesis in Met-dependent cells and a postulation that Met-dependent cells are deficient in utilization of endogenously synthesized Met as opposed to exogenously supplied Met. To answer the critical question of what biochemical reaction(s) requires preformed Met in Met-dependent cels, we labeled cells with Met-free [35S]Hcy or [35S]Met and determined the levels of Met, S-adenosylmethionine (AdoMet), and S-adenosylhomocysteine (AdoHcy). We report here experiments that demonstrate that Met-dependent cells synthesize a normal amount of endogenously synthesized Met and are deficient in utilizing this Met for AdoMet synthesis. In contrast, exogenously supplied Met is utilized normally for AdoMet biosynthesis. The ratio of AdoMet to AdoHcy is low in Met-dependent cells growing in Met-Hcy+ medium but is normal in Met+Hcy- medium. We determined that the low AdoMet/AdoHcy ratio probably limits growth of Met-dependent cells in Met-Hcy+ medium.
Cystine accumulation and clearance by normal and cystinotic leukocytes exposed to cystine dimethyl ester.
Authors: Steinherz R , Tietze F , Gahl W A , Triche T J , Chiang H , Modesti A , Schulman J D
Published: May 2006

Upon exposure to 0.25 mM cystine dimethyl ester, normal and cystinotic leukocytes accumulate substantially more intracellular cystine than is present endogenously in cystinotic cells. Leukocytes loaded by exposure to cystine dimethyl ester may have abnormally lucent and distended lysosomes, and the cystine is compartmentalized within the granular fraction of the cells. After the cells are exposed to cystine dimethyl ester, cystine clearance from normal leukocytes is much faster than from cystinotic cells. The ratios of labeled cysteine-N-ethylmaleimide to cystine are also greater in normals than in cystinotics 60 min after termination of loading. No overlap in ranges of cystine clearance half-times or cystine-N-ethylmaleimide to cystine ratios was observed in normal compared to cystinotic leukocytes. Limited experiments with fibroblasts exposed to cystine dimethyl ester suggest a correspondingly prolonged cystine clearance for cystinotic cells. These experiments provide evidence for defective clearance of cystine from cystinotic lysosomes in situ.
Molecular cloning of cDNA coding for the gamma subunit of Torpedo acetylcholine receptor.
Authors: Ballivet M , Patrick J , Lee J , Heinemann S
Published: May 2006

From the electric organ of Torpedo californica, we purified mRNA that, when translated in vitro, produces polypeptides immunoprecipitable by antibodies against purified acetylcholine receptor. A novel cloning system [Okayama, H. & Berg, P. (1982) Mol. Cell. Biol. 2, 161-170] was used to produce a cDNA library from this mRNA. This library contained clones with receptor sequences identified by differential hybridization and hybridization-selection. We describe a clone of 2,030 base pairs with sequences appropriate for the amino-terminal amino acids of the gamma subunit of acetylcholine receptor. This clone contains 82 bases 5' of the codon for the amino-terminal amino acid of the mature protein. A portion of this sequence codes for a methionine followed by a 16-amino acid polypeptide that is contiguous to the amino-terminal amino acid of the mature protein and that has the characteristics of a leader peptide. The cDNA insert hybridizes to a 2,100-base RNA present in electric organ but not in the brain of T. californica.
Chromosomal mapping of the simian sarcoma virus onc gene analogue in human cells.
Authors: Swan D C , McBride O W , Robbins K C , Keithley D A , Reddy E P , Aaronson S A
Published: May 2006

The primate cell-derived transforming gene (v-sis) of simian sarcoma virus (SSV) is represented as a single copy marker within cellular DNAs of mammalian species including human. The human analogue of v-sis can be distinguished from its rodent counterparts by Southern blotting analysis of EcoRI-restricted DNAs. By testing for the presence of the human v-sis-related fragment, c-sis (human), in somatic cell hybrids possessing varying numbers of human chromosome, as well as in segregants of such hybrids, it was possible to assign c-sis to human chromosome 22.
Structural basis for receptor binding heterogeneity of apolipoprotein E from type III hyperlipoproteinemic subjects.
Authors: Rall S C , Weisgraber K H , Innerarity T L , Mahley R W
Published: May 2006

The three major isoforms of human apolipoprotein E (apo-E2, -E3, and -E4) are coded for by three alleles (epsilon 2, epsilon 3, and epsilon 4) which have a common genetic locus. Previously, we demonstrated that E2, E3, and E4 differ in primary structure from one another at two substitution sites, site A (residue 112) and site B (residue 158). At sites A/B, apo-E2, -E3, and -E4 contain cysteine/cysteine, cysteine/arginine, and arginine/arginine, respectively. We demonstrated that the substitution of cysteine for arginine at site B is at least partly responsible for the defective binding of apo-E2 to human fibroblast low density lipoprotein receptors, compared to the normal binding activity of apo-E3 or -E4. Subjects with the genetic disorder type III hyperlipoproteinemia are phenotypically homozygous for apo-E2, but the binding activity of apo-E to the fibroblast receptor differs considerably from one type III individual to another. We therefore undertook a partial comparative sequence analysis of apo-E2 from three type III subjects whose apo-E displayed this heterogeneity. The subject with the poorest binding apo-E2 was genotypically homozygous for an apo-E allele (epsilon 2); cysteine was found at sites A and B. The subject with the most active apo-E2 was genotypically homozygous for an apo-E allele (epsilon 2); cystine was found at site A and at a new site (site C, residue 145). The epsilon 2 allele specifies a protein that has arginine at site B (residue 158); the epsilon 2 allele specifies a protein that has arginine at site C (residue 145). Therefore, the two alleles differ from one another by cysteine/arginine interchanges at two positions, sites B and C. The third subject, whose apo-E2 displayed binding activity intermediate between the activities of the other two, was genotypically heterozygous, having one epsilon 2 allele and one epsilon 2 allele. The intermediate binding activity of apo-E2 from this subject resulted from having a mixture of severely defective apo-E (specified by epsilon 2) and slightly defective apo-E (specified by epsilon 2).
Chromosomal assignment of the human homologues of feline sarcoma virus and avian myeloblastosis virus onc genes.
Authors: Dalla-Favera R , Franchini G , Martinotti S , Wong-Staal F , Gallo R C , Croce C M
Published: May 2006

Retroviral transforming genes, v-onc genes, are derived from normal cellular sequences that are called cellular onc (c-onc) genes. DNA from mouse-human somatic cell hybrids that have selectively lost human chromosomes was used in Southern blots to map the chromosomal location of two human onc genes. Cloned human homologues of retroviral onc genes were used as probes. Because the human c-fes gene, which is homologous to feline sarcoma virus, segregates concordantly with human chromosome 15, and the human c-myb gene, which is homologous to avian myeloblastosis virus onc genes, segregates concordantly with human chromosome 6, we have assigned the c-fes and the c-myb genes to human chromosomes 15 and 6, respectively. Nonrandom chromosomal defects involving these human chromosomes have been observed in neoplasms. These studies should be valuable in determining whether specific rearrangements involving these chromosomes result in the abnormal expression of these onc genes in human malignancies.
Human amniotic fluid cells grown in a hormone-supplemented medium: suitability for prenatal diagnosis.
Authors: Chang H C , Jones O W , Masui H
Published: May 2006

A new supplemented medium has been developed to improve human amniotic fluid cell growth and to reduce the dependence on exogenously added serum. The medium consists of a mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium supplemented with Hepes, antibiotics, and 10 growth-promoting factors at 4% fetal bovine serum. Good clonal growth is achieved consistently in 8--13 days and is associated with large numbers of metaphase cells. Primary clones may be analyzed directly, thereby reducing difficulty with interpretation of chromosomal mosaicism. This medium could also be used for cultivation of fetal solid tissues and peripheral blood cultures of lymphocytes.
Opiate antagonistic properties of an octapeptide somatostatin analog.
Authors: Maurer R , Gaehwiler B H , Buescher H H , Hill R C , Roemer D
Published: May 2006

The somatostatin analog D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-NH-CH(CH2OH)CHOHCH3 (SMS 201-995) displaces [3h[naloxone from its binding sites (IC50, 38 +/- 60 nM), being more than 200 times more potent than somatostatin. As measured by the difference between [3H]dihydromorphine, [3H][D-Ala2,D-Leu5]enkephalin, and (-)-[3H]bremazocine binding, SMS 201-995 appears to be highly specific for the opiate mu binding site. Electrophysiological data from hippocampal cultures and results from animal studies (tail flick, mydriasis) demonstrate the opiate antagonistic properties of SMS 201-995. SMS 201-995 is an opiate mu antagonist with a peptide structure. That this property is displayed by a somatostatin analog is somewhat unexpected.
Protease La from Escherichia coli hydrolyzes ATP and proteins in a linked fashion.
Authors: Waxman L , Goldberg A L
Published: May 2006

The energy requirement for protein breakdown in Escherichia coli results from an ATP requirement for the function of protease La, the product of the lon gene. This novel serine protease contains an ATPase activity that is essential for proteolysis. ATP and protein hydrolysis show the same Km for ATP (30-40 muM) and are affected similarly by various inhibitors, activators, and ATP analogs. Vanadate inhibited ATP cleavage and caused a proportionate reduction in casein hydrolysis, and inhibitors of serine proteases reduced ATP cleavage. Thus, ATP and protein hydrolysis appear to be linked stoichiometrically. Furthermore, ATP hydrolysis is stimulated two- to threefold by polypeptides that are substrates for the protease (casein, glucagon) but not by nonhydrolyzed polypeptides (insulin, RNase). Unlike hemoglobin or native albumin, globin and denatured albumin stimulated ATP hydrolysis and were substrates for proteolysis. It is suggested that the stimulation of ATP hydrolysis by potential substrates triggers activation of the proteolytic function.
Isolation of a selenium-containing thiolase from Clostridium kluyveri: identification of the selenium moiety as selenomethionine.
Authors: Hartmanis M G , Stadtman T C
Published: May 2006

Clostridium kluyveri grown in the presence of 1 muM Na2(75)SeO3 produces a thiolase that copurifies with 75Se. Based on several criteria, the selenium moiety in this protein is selenomethionine. The 75Se-labeled amino acid in acid hydrolysates of the radioactive protein cochromatographed with authentic selenomethionine on an amino acid analyzer and on TLC plates in acidic and basic solvents. Incubation with S-adenosylmethionine synthetase and ATP converted the 75Se-labeled amino acid to a radioactive basic product that was indistinguishable from authentic Se-adenosylselenomethionine by ion exchange and TLC. The native selenoenzyme, Mr 155,000-158,000, is composed of four subunits of Mr 38,000-40,000. Thiolase of similar molecular weight that is less acidic and lacks selenium is also produced by C. kluyveri. The factors that control the relative levels of the two enzymes in the cell have not been identified.
The accuracy of protein biosynthesis is limited by its speed: high fidelity selection by ribosomes of aminoacyl-tRNA ternary complexes containing GTP[gamma S]
Authors: Thompson R C , Karim A M
Published: May 2006

Guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] ) forms a stable ternary complex with polypeptide chain elongation factor Tu (EF-Tu) and aminoacyl-tRNA, and this complex binds rapidly and tightly to a properly programmed ribosome. However, the rate constant for the subsequent hydrolysis of the beta-gamma pyrophosphate bond (3.9 X 10(-3) s-1 at 5 degrees C) is less than 1/2,500th of that for the analogous reaction of GTP. We have taken advantage of this low rate to determine the rate constant for dissociation of the complex of poly(U)-programed ribosomes, EF-Tu, Phe-tRNAPhe, and GTP[gamma S] (2.7 X 10(-3) s-1) and the second-order rate constant for formation of this complex (3.3 X 10(6) M-1 s-1). Therefore, the Kd of the complex may be calculated to be 8.2 X 10(-10) M. An analogous near-cognate complex with Leu-tRNA2Leu in place of Phe-tRNAPhe has been determined by equilibrium methods to have a Kd greater than 1.7 X 10(-6) M. These results indicate that under equilibrium conditions the ribosome can distinguish cognate and near-cognate ternary complexes with great accuracy. Therefore, its failure to show this high specificity with the physiological ternary complexes containing GTP is due to the speed of GTP hydrolysis being similar to the speed of dissociation of the near-cognate complex. The low specificity of the physiological reaction is corrected by subsequent proofreading. The results reported here suggest that proofreading is necessary not simply for high accuracy but for the combination of speed and accuracy required in protein biosynthesis.
Glucocorticoids increase the Na+-H+ exchange and decrease the Na+ gradient-dependent phosphate-uptake systems in renal brush border membrane vesicles.
Authors: Freiberg J M , Kinsella J , Sacktor B
Published: May 2006

The glucocorticoid dexamethasone, but not the mineralocorticoid aldosterone, increased amiloride-sensitive Na+-H+ exchange activity in rat proximal tubule brush border vesicles. Na+ uptake, independent of amiloride, was not affected. The glucocorticoid decreased the Na+ gradient-dependent phosphate uptake. Uptake in the absence of a Na+ gradient was not inhibited. Dexamethasone did not affect the Na+ gradient-dependent D-glucose uptake. These findings are consistent with the effects of glucocorticoids in stimulating acid secretion and causing phosphaturia in man and animals and may identify the locus of action and suggest the mechanisms by which the hormones act.
Common 82-nucleotide sequence unique to brain RNA.
Authors: Sutcliffe J G , Milner R J , Bloom F E , Lerner R A
Published: May 2006

Several randomly selected cDNA clones made from rat brain polyA+RNA have unusual properties. Although the cDNA inserts are 500-1,250 base pairs long, they hybridize to a 160-nucleotide RNA species that is present in brain but not in liver or kidney. Nucleotide sequence studies of two of the clones and hybridization studies show that a common 82-nucleotide sequence is responsible for the hybridization. The same sequence is located in the second intron of a rat growth hormone gene. These studies suggest that signals called "ID sequences" may be carried in the introns of genes and prescribe their tissue-specific expression.
Kinetic evidence suggesting two mechanisms for iodothyronine 5'-deiodination in rat cerebral cortex.
Authors: Visser T J , Leonard J L , Kaplan M M , Larsen P R
Published: May 2006

Enzymatic 5'-deiodination of 3,3',5'-triiodothyronine (rT3) and 3,3',5,5'-tetraiodothyronine (thyroxine, T4) was studied in microsomal preparations of rat cerebral cortex. Evidence was obtained for the existence of two thiol-dependent 5'-deiodinase entities. One of these predominates in tissue from euthyroid and long-term hypothyroid rats, is specific for rT3, follows "ping-pong" kinetics with dithiothreitol as the cosubstrate, and is inhibited by propylthiouracil (PrSUra) and iodoacetate. Inhibition by PrSUra is uncompetitive with rT3 and competitive with dithiothreitol. These properties are shared with the 5'-deiodinase activity of liver and kidney. The activity of a second type of 5'-deiodinase is highest in cerebral cortex from short-term hypothyroid rats, prefers T4 to rT3 as the substrate, is insensitive to PrSUra and iodoacetate, and follows "sequential" reaction kinetics. A similar PrSUra-insensitive 5'-deiodinase activity is also found in pituitary but is not detectable in liver and kidney; it seems, therefore, characteristic of tissues in which local T4 to 3,3',5-triiodothyronine (T3) conversion supplies a major portion of the total intracellular T3.
Calculation of the three-dimensional structure of the membrane-bound portion of melittin from its amino acid sequence.
Authors: Pincus M R , Klausner R D , Scheraga H A
Published: May 2006

The structure of the NH2-terminal, 20-residue membrane-bound portion of melittin has been computed with empirical energies (ECEPP, Empirical Conformational Energy Program for Peptides). First, a search was made for long stretches of nonpolar residues. Then, the low-energy conformations of these segments were built up by combining successively the low-energy conformations of their component di- and tripeptides. The minimum-energy conformations of each of these component peptides used in this buildup process were selected so that each had a distinct backbone conformation; these distinct backbone conformations were designated as nondegenerate minima. Structured segments (i.e., those with only a few low-energy conformations) resulting from this process were identified and used as nucleation sites for building up larger structures by adding adjacent peptide segments. At each stage, the energy of each structure was minimized. Only two low-energy structures were found, both of which were alpha-helical from Gly-1 to Thr-10 and from Pro-14 to Ile-20. In one of them, the first helix continues through Thr-11-Gly-12; in the other, there is a bend at Thr-11-Gly-12. However, because of compensating changes in the dihedral angles, both structures are very similar. The calculated structures seem to agree well with experimental data.
Dynamics and time-averaged chemical potential of proteins: importance in oligomer association.
Authors: Xu G , Weber G
Published: May 2006

The chemical potential of a protein is a time-averaged quantity whose value depends upon the fractions of time spent in the different conformations and therefore upon the protein dynamics. If the monomer involved in an association equilibrium undergoes unfolding limited by its lifetime, its chemical potential as well as that of the associated form will not be constant and the free energy of association will be a diminishing function of the degree of dissociation. For a unique free energy of association, the logarithm of the protein concentration must change by 2.86 units to increase the degree of dissociation from 0.1 to 0.9. The dissociation of enolase appears to take place over a significantly smaller range (1.7 units), and dansyl conjugates of enolase show an even narrower range (0.9 unit). A simple descriptive theory is developed, and this shows that the values observed are explained by a difference in free energy of 1-3 kcal/mol (1 cal = 4.18 J) between the conformations present at negligible and almost complete dissociation.
Somatic cell parasitism and the evolution of somatic tissue compatibility.
Authors: Buss L W
Published: May 2006

Selection pressures proposed to account for the convergent evolution of self/not-self recognition systems in lower organisms include defense against microbial parasites and somatic cell variants. Direct support for the existence of somatic cell parasites in natural populations has been lacking. I here report the occurrence of a somatic cell parasite in the cellular slime mold Dictyostelium mucoroides and discuss the implications of this phenomenon to the evolution of mechanisms of somatic tissue compatibility.
Single-copy sequence hybridizes to polymorphic and homologous loci on human X and Y chromosomes.
Authors: Page D , de Martinville B , Barker D , Wyman A , White R , Francke U , Botstein D
Published: May 2006

Use of a 4.5-kilobase-pair (kb) segment of single-copy DNA from a human genomic library as a hybridization probe of genomic human DNAs revealed allelic Taq I restriction fragments 10.6, 11.8, and 14.6 kb long. Among 12 unrelated individuals, all 6 males exhibited the 14.6-kb fragment in addition to one of the other fragments. Three of the females displayed 10.6- and 11.8-kb fragments, and the other three displayed only one fragment length; none had the 14.6-kb fragment. Hybridization of this probe to Taq I-digested DNAs from human-rodent hybrid cell lines (which have partial complements of human chromosomes) demonstrated segregation of the 14.6-kb fragment with the human Y chromosome and segregation of the 10.6- and 11.8-kb fragments with the human X chromosome. Furthermore, hybridization of this probe to Taq I-digested DNAs from 48 members of a single kindred revealed Y-linked inheritance of the 14.6-kb fragment and X-linked inheritance of the 10.6- and 11.8-kb fragments. These experiments demonstrate homology between single-copy sequences on the human X and Y chromosomes.
Map of distamycin, netropsin, and actinomycin binding sites on heterogeneous DNA: DNA cleavage-inhibition patterns with methidiumpropyl-EDTA.Fe(II).
Authors: Van Dyke M W , Hertzberg R P , Dervan P B
Published: May 2006

We report a direct technique for determining the binding sites of small molecules on naturally occurring heterogeneous DNA. Methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II) cleaves double helical DNA with low sequence specificity. Using a combination of MPE.Fe(II) cleavage of drug-protected DNA fragments and Maxam-Gilbert gel methods of sequence analysis, we have determined the preferred binding sites on a Rsa I-EcoRI restriction fragment from pBR322 for the intercalator actinomycin D and the minor groove binders netropsin and distamycin A. Netropsin and distamycin A gave identical DNA cleavage-inhibition patterns and bound preferentially to A+T-rich regions with a minimal protected site of four base pairs. We were able to observe the effect of increasing concentration on site selection by netropsin and distamycin A. Actinomycin D afforded a completely different cleavage-inhibition pattern, with 4- to 16-base-pair-long protected regions centered around one or more G.C base pairs.