bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of Schistosoma mansoni egg-induced granuloma formation: I-J restriction of T cell-mediated suppression in a chronic parasitic infection.
Authors: Green W F , Colley D G
Published: May 2006

Newly formed hepatic granulomas around Schistosoma mansoni eggs become progressively smaller during the chronic (greater than or equal to 15 weeks after infection) phase of the disease. This reduction in granuloma size, termed "modulation," is known to be caused in part by a T lymphocyte that can adoptively transfer modulation to 6-week-infected mice. The present study examines a possible role for the I-J locus in regulating the suppressor T lymphocyte aspects of modulation. Adoptive transfer between congeneic B10.A(3R) and B10.A(5R) mice (differing at the I-J locus) indicated that optimal suppression is dependent upon homology at the I-J locus. In vivo treatment of chronically infected mice with microliter amounts of antiserum specific for the recipient's I-J determinant blocked modulation during chronic infection and prevented adoptive transfer of suppression to 6-week-infected mice. The in vivo regimen of anti-I-J had no effect on anti-schistosomal egg antigen titers during chronic infection. These results demonstrate an I-J restriction for suppression. It appears that the suppressor T lymphocyte circuit responsible for this aspect of modulation requires an I-J positive lymphocyte.
Multiple biologic activities of a cloned inducer T-cell population.
Authors: Nabel G , Greenberger J S , Sakakeeny M A , Cantor H
Published: May 2006

The mouse T-cell clone Ly1+2(-)/9, belonging to the Ly1 set, displays the following functions in vitro: (i) augmentation of immunoglobulin output by B cells; (ii) stimulation of bone marrow cells to produce colonies composed of granulocytes, macrophages, or both; and (iii) proliferative stimulation of T-cell clones belonging to other Ly sets. These functions are induced by Ly1+2(-)/9 cells themselves and by supernatants of Ly1+2(-)/9 cultures and are not evinced by tested clones belonging to other Ly sets. The agent or agents responsible for colony formation and for B-cell stimulation had an apparent molecular weight of 45,000-50,000 and could not be physically separated. The T-cell stimulating agent(s) had an apparent molecular weight of 30,000 and could be separated from the agent(s) that acts upon colony formation and B cells. Thus, clone LY1+2(-)/9 produces at least two soluble products that induce or augment activities of at least three other differently programmed cell sets.
Complete amino acid sequence of bovine thymosin beta 4: a thymic hormone that induces terminal deoxynucleotidyl transferase activity in thymocyte populations.
Authors: Low T L , Hu S K , Goldstein A L
Published: May 2006

The amino acid sequence of thymosin beta 4, a polypeptide isolated from calf thymus, was determined. Thymosin beta 4 is composed of 43 amino acid residues and has a molecular weight of 4982 and an isoelectric point of 5.1. The NH2 terminus of the peptide is blocked by an acetyl group. This molecule induces expression of terminal deoxynucleotidyl transferase (DNA nucleotidylexotransferase, EC 2.7.7.31) in transferase-negative murine thymocytes in vivo and in citro. Thus, it appears that thymosin beta 4 acts on lymphoid stem cells and may control the early stages of the maturation process of thymus-dependent lymphocytes. This peptide is one of several present in thymosin fraction 5 that participates in the regulation, differentiation, and function of thymus-dependent thymocytes.
Two allelic forms of mouse beta 2-microglobulin.
Authors: Robinson P J , Graf L , Sege K
Published: May 2006

Two allelic forms of mouse beta 2-microglobulin (beta 2m), the small polypeptide chain of H-2 histocompatibility antigens, have been identified. The two forms can be distinguished by NaDodSO4/polyacrylamide gel electrophoresis. All inbred mouse strains express a single beta 2m isotype. Mice heterozygous for beta 2m synthesize both forms, showing codominant expression of beta 2m alleles. In mice heterozygous for both H-2 and beta 3m, individual H-2 histocompatibility antigens associate with both beta 2m forms. Preliminary structural studies indicate differences in peptide composition between the two forms.
Inhibition of tumor growth, vascularization, and collagenolysis in the rabbit cornea by medroxyprogesterone.
Authors: Gross J , Azizkhan R G , Biswas C , Bruns R R , Hsieh D S , Folkman J
Published: May 2006

Medroxyprogesterone, dexamethasone, or cortisone, locally applied in sustained release polymer to rabbit V2 carcinoma implanted in the rabbit cornea, blocked neovascularization and three-dimensional growth of the tumor. These hormones similarly prevented the vascular proliferative response to implants in the rabbit cornea of mouse B-16 melanoma and also the response to implants of polymer containing tumor extract with angiogenesis activity. The inhibitory responses were accompanied by considerable reduction in collagenolytic activity released into culture medium by explants of the two tumors and of the corneal region containing angiogenic hepatoma extract. Morphologic studies revealed extensive three-dimensional disruption of the compact laminated collagenous structure of the cornea by untreated V2 carcinoma. In the presence of hormone the tumor grew slowly as a noninvasive two-dimensional plaque limited to the narrow region of the insertion pocket in the cornea, with no obvious disturbance of structure elsewhere. Cortisone was much les effective than medroxyprogesterone or dexamethasone. Testosterone and estradiol had no effect on the three measured properties. The data suggest that local hormonal interference with neovascularization, collagenase production, and tumor growth can prevent neoplastic invasion and destruction of a dense collagenous connective tissue.
Localization of 25-hydroxyvitamin D3 1 alpha-hydroxylase and 24-hydroxylase along the rat nephron.
Authors: Kawashima H , Torikai S , Kurokawa K
Published: May 2006

Defined nephron segments were microdissected from the kidney of vitamin D-deficient rats, normal rats, and normal rats treated with 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25-(OH)2D3]. Tubule segments were incubated with 3H]labeled 25-hydroxyvitamin D3 and the rates of production of 3H]labeled 1 alpha, 25-(OH)2D3 and 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] were determined. Nephron segments tested include the glomerulus, proximal convoluted tubules (PCT), proximal straight tubules (PST), medullary and cortical thick ascending limbs of Henle's loop, distal tubules, and collecting tubules. Production of 1 alpha, 25-(OH)2D3 was detected only in PCT of vitamin D-deficient rats (mean +/- SEM, 0.70 +/- 0.05 fmol/mm per hr); the value decreased to 0.11 +/- 0.05 after parathyroidectomy. By contrast, significant 24,25-(OH)2D3 production occurred in PCT of normal rats (0.23 +/- 0.05 fmol/mm per hr). After administration of 1 alpha, 25-(OH)2D3 to normal rats, the rate of production of 24,25-(OH)2D3 in PCT increased to 0.64 +/- 0.06 fmol/mm per hr and also became apparent in PST (1.07 +/- 0.21). The rates of production of 1 alpha, 25-(OH)2D3 and 24,25-(OH)2D3 in these nephron segments were linear with tubule length over a wide range of lengths per incubation and with the incubation time. The results define the localization of 25-hydroxyvitamin D3 1 alpha-hydroxylase and 24-hydroxylase along the rat nephron: PCT is capable of producing both 1 alpha, 25-(OH)2D3 and 24,25-(OH)2D3 and PST can produce 24,25-(OH)2D3. the use of defined nephron segments may be useful for study of the distribution and regulation of 25-hydroxyvitamin D3 hydroxylases in the kidney.
Na+-channel-associated scorpion toxin receptor sites as probes for neuronal evolution in vivo and in vitro.
Authors: Berwald-Netter Y , Martin-Moutot N , Koulakoff A , Couraud F
Published: May 2006

Purified neurotoxin II of the scorpion Androctonus australis Hector (ScTx) has previously been shown to bind specifically to the Na+-ionophore-associated, voltage-sensitive receptor sites of excitable cells. We have conducted binding studies, using high-specific-activity 125I-labeled ScTx, to detect and quantify the Na+-channel receptors on cells of the developing fetal mouse brain. In vivo, the onset of detectable specific binding is at 12 fetal days. The rate of receptor appearance is initially slow but increases sharply as of the 16th day of mouse ontogenesis. The mean number of receptors at 12 and 19 days is 120 and 20,000 per cell, respectively (i.e., 0.5 and 80 per square micrometer). When corrected for the fraction of cell population corresponding to putative neuroblasts and neurons, identified by immunofluorescence as tetanus toxin binding cells, these values are, respectively, 1040 and 33,900 ScTx receptors per tetanus toxin binding cell or 4.2 and 136 per square micrometer. At all stages, the toxin binds to a single class of noninteracting sites; Kd = 0.1-0.5 nM. Similar findings in terms of ScTx-receptor properties and quantitative evolution were obtained in vitro. Specific 125I-labeled ScTx binding the presence of tetanus toxin binding cells. In cultures of central nervous system glia without neurons, only nonspecific low-level ScTx binding was detected. These results suggest that the high-affinity scorpion toxin receptors may be used as quantitative markers of neuronal differentiation.
Specific monosaccharide inhibition of active sodium channels in neuroblastoma cells.
Authors: Giovanni M Y , Kessel D , Glick M C
Published: May 2006

L-Fucose and D-galactose in low concentrations (0.27 or 2.7 mM) inhibited the induction of active Na+ channels in mouse and human neuroblastoma cells when the monosaccharides were added to the culture medium for 4 days with the inducing agent dimethyl sulfoxide. Active Na+ ionophores were determined by measurement of the toxin-stimulated efflux of 86Rb from the cells. At the same time, the amount of a radioactive glycoprotein (Mr 200,000), which was shown previously to be associated with neurite and membrane preparations from cells with active Na+ channels, was decreased. Cell growth and viability were not affected. The nonphysiological isomer D-fucose or the addition of D-glucose in the same concentration did not inhibit differentiation. Vibrio cholerae neuraminidase, added to the cells prior to the stimulation of 86Rb efflux by veratridine and scorpion venom, was inhibitory. The implications of these findings, which suggest a key role for glycoproteins in at least a portion of the excitability process, are discussed.
Dynorphin immunocytochemical localization in brain and peripheral nervous system: preliminary studies.
Authors: Watson S J , Akil H , Ghazarossian V E , Goldstein A
Published: May 2006

Using antisera specific for the opioid peptide dynorphin, we have carried out immunocytochemical studies of the distribution in rat brain and periphery. In the central nervous system, cells that stain positively for dynorphin are found in the supraoptic nucleus, with less-well-stained cells in the paraventricular nucleus of the hypothalamus. Few positive fibers were detected in brain, suggesting problems with fixation and preservation of antigenicity. In pituitary no staining was seen in the anterior and intermediate lobes but heavy staining was detected in the posterior lobe. In the guinea pig, adrenal chromaffin cells stained with dynorphin antisera. Staining of these cells could be blocked with excess of dynorphin-(1-13) or either enkephalin. Radioimmunoassays revealed a great excess of the enkephalins in the adrenal, suggesting cross competition between dynorphin antiserum and adrenal medullary enkephalin. Finally, the dynorphin antiserum stained a complex of fibers in guinea pig ileum. Staining of these fibers could be blocked by moderate amounts of enkephalin as well as by smaller amounts of dynorphin-(1-13). We conclude that in some places (brain and pituitary) dynorphin exists separately from leucine-enkephalin. In other parts of brain and in the periphery the relationship between dynorphin and the enkephalins is very complex and requires further study and improved antisera.
Membrane lipid heterogeneity associated with acetylcholine receptor particle aggregates in Xenopus embryonic muscle cells.
Authors: Bridgman P C , Nakajima Y
Published: May 2006

Filipin, digitonin, and saponin react with membrane cholesterol to produce unique membrane alterations (sterol-specific complexes) that are easily discernible in freeze-fracture replicas. We have treated both noninnervated and innervated Xenopus embryonic muscle cells in culture with these agents. Freeze-fracture of these treated muscle cells showed that most areas of the muscle plasma membrane contain sterol-specific complexes (19- to 40-nm protuberances and dimples with filipin, a scalloped appearance with digitonin, or an irregular, rough appearance with saponin). However, these complexes were virtually absent from membrane areas of junctional and nonjunctional aggregates of acetylcholine receptor particles. This result suggests that the membrane matrix of these aggregates is low in cholesterol and that this membrane lipid heterogeneity may be linked to the mechanisms involved in their formation and stabilization on muscle cells in culture.
Cell surface modulation of gene expression in brain cells by down regulation of glucocorticoid receptors
Authors: McGinnis James F. , De Vellis Jean
Published: May 2006

The concentration of glycerol-3-phosphate dehydrogenase (GPDH;
sn
-glycerol-3-phosphate:NAD
+
2-oxidoreductase, EC 1.1.1.8) had previously been determined to be regulated by glucocorticoids in rat brain cells
in vivo
and in cell culture. We now demonstrate that concanavalin A (Con A) can inhibit the induction of GPDH in dose-dependent manner in C6 rat glioma cells and in primary cultures of rat brain oligodendrocytes. Con A is not cytotoxic, because its effect can be prevented or reversed by α-methyl mannoside. The inhibition specifically prevents the appearance of new molecules of GPDH, although Con A does not significantly inhibit protein synthesis in these cells, nor does it affect the activity of another soluble enzyme, lactate dehydrogenase. The ability to block enzyme induction is not limited to Con A, because other lectins also inhibit induction, with
Ricinus communis
agglutinin 60 being the most potent (50% inhibition of induction at 0.0083 μM) and wheat germ agglutinin being the least potent (50% inhibition of induction at 1.2 μM). The molecular mechanism by which Con A inhibits GPDH induction appears to be by the “down regulation” of the cytoplasmic glucocorticoid receptors, because exposure to Con A results in the loss of more than 90% of the receptor activity. Con A does not inhibit the receptor assay and no direct interaction between the receptor and Con A could be demonstrated. This down regulation is not tumor cell specific and appears to be a general phenomenon, because it occurs in normal oligodendrocytes and even in normal astrocytes (a cell type in which the gene for GPDH is not expressed). The down regulation of glucocorticoid receptors in normal brain cells suggests two important corollaries. First, it demonstrates the existence of a rate-limiting step controlling the glucocorticoid-dependent gene expression in brain cells and possibly represents a regulatory site common to all glucocorticoid target cells. Second, it suggests that the response to glucocorticoids of oligodendrocytes and astrocytes can be regulated
in vivo
by cell surface contact with endogenous lectins, neighboring cells, or both.
Nonbonded interaction potentials in methyl-substituted butanes, obtained from high-energy overtone spectra
Authors: Mohammadi M. Ali , Henry Bryan R.
Published: May 2006

The observed decrease in local mode anharmonicity with increasing methyl substitution in butane is related to a change in potential energy along a methyl CH-stretching coordinate. The potential energy change is modeled by Lennard-Jones 6-12 and Morse potentials and the resultant potential parameters are discussed.
Developmental regulation of secretory protein synthesis in rat seminal vesicle.
Authors: Kistler M K , Ostrowski M C , Kistler W S
Published: May 2006

This paper reports a developmental study of one of the major secretory proteins (SVS IV) of the rat seminal vesicle. Measured by radioimmunoassay, SVS IV is detectable even in very young rats (5 days old) but does not begin to accumulate in massive amounts until animals reach about 35 days of age. We have compared SVS IV synthesis by minced tissue from rats undergoing sexual maturation (25-60 days of age) with levels of translatable SVS IV mRNA present. Between 25 and 60 days of age, the proportion of newly synthesized protein devoted to SVS IV by tissue minces increased from 0.8% to 20%. In contrast, the proportion of total RNA devoted to translatable SVS IV mRNA was already high in the immature glands and increased less than 4-fold during development. These results seem to indicate that factors other than the presence of potentially translatable message regulate the rate of net synthesis of SVS IV during seminal vesicle maturation.
Catenation and knotting of duplex DNA by type 1 topoisomerases: a mechanistic parallel with type 2 topoisomerases.
Authors: Brown P O , Cozzarelli N R
Published: May 2006

Escherichia coli omega protein, a type 1 topoisomerase, can catenate and knot duplex DNA circles. Previously, these activities were thought to be limited to type 2 topoisomerases. Catenation by omega requires a nick in one of the participating molecules, but it is not necessary that both be nicked. Catenation does not depend on sequence homology and is greatly stimulated by DNA-condensing agents such as spermidine. A eukaryotic type 1 topoisomerase can also interlock duplex DNA circles. These activities cannot easily be explained by the widely held topoisomerase model in which a reversible nick in DNA allows free rotation about the unbroken strand. We suggest instead passage of a DNA segment though a transient enzyme-bridged break in a single DNA strand. This is analogous to the sign inversion mechanism of the type 2 topoisomerases, and thus expresses an essential mechanistic unity among topoisomerases. As expected for relaxation by a single-strand passage, omega changes the linking number of DNA in steps of 1.
Translational stability of native and deadenylylated rabbit globin mRNA injected into HeLa cells.
Authors: Huez G , Bruck C , Cleuter Y
Published: May 2006

HeLa human cells were injected with a natural mixture of rabbit alpha and beta globin mRNA. They were incubated for 6 hr with [35S]methionine either immediately after injection or 20 hr later. The labeled proteins in the injected cells were analyzed by fluorography of two-dimensional electrophoresis gels. By using this procedure, it was possible to show that, during the first few hours after injection, both alpha and beta globin molecules are synthesized with an alpha to beta ratio approximately equal to 0.6. The rate of synthesis of alpha globin decreased significantly faster than that of beta globin over a 26-hr period after injection of the two mRNAs. It thus seems that two messenger RNAs coding for closely related polypeptides possess a markedly different translational stability. When deadenylylated rabbit globin mRNAs were injected into HeLa cells, no globin synthesis could be detected by the techniques used. We conclude that the translational half-life of mRNAs lacking poly(A) is very short in these cells. It is thus clear that the poly(A) segment is required to ensure stability to globin mRNA in somatic cells as in Xenopus oocytes.
Bacterial bioluminescence: Spectral study of the emitters in the
in vitro
reaction
Authors: Matheson I. B. C. , Lee John , Müller Franz
Published: May 2006

Transient fluorescent species are observed in the bioluminescent reactions of three reduced flavin mononucleotides with aliphatic aldehydes and oxygen, catalyzed by bacterial luciferase. In each case the fluorescence spectral distribution is similar to that of the bioluminescence but is readily distinguishable from it on the basis of a significantly greater signal strength. The corrected bioluminescence maxima using
Beneckea harveyi
luciferase are 479 nm (iso-FMNH
2
), 490 nm (FMNH
2
), and 560 nm (2-thio-FMNH
2
). In an ethanol glass at 77 K, 2-thioriboflavin is fluorescent (ϕ

F

= 0.03, λ
max
= 562 nm). These results are interpreted by a sensitized chemiluminescence mechanism in which the flavins bound to luciferase act as acceptors of excitation energy. For 2-thio-FMNH
2
, this acceptor species appears to be the oxidized 2-thio-FMN on the basis of the spectral evidence, whereas for the other flavins, some form of reduced species is a more likely candidate.
Picosecond resonance Raman spectroscopic evidence for excited-state spin conversion in carbonmonoxy-hemoglobin photolysis
Authors: Terner James , Stong John D. , Spiro Thomas G. , Nagumo Mark , Nicol Malcolm , El-Sayed Mostafa A.
Published: May 2006

The structure of the carbonmonoxy-hemoglobin (COHb) photoproduct has been studied on the picosecond time scale with resonance Raman spectroscopy, by tightly focusing the 30-ps pulses of a synchronously pumped mode-locked cavitydumped dye laser on a jet stream of COHb solution. The spectrum of the photoproduct is similar to that of deoxy Hb, but the frequencies 1603 cm
-1
(depolarized), 1552 cm
-1
(anomalously polarized), and 1542 cm
-1
(depolarized) are 2-4 cm
-1
lower than those of deoxy Hb. Similar low frequencies are observed for a species believed to be the bis-tetrahydrofuran adduct of Fe
II
octaethylporphyrin, containing in-plane high-spin Fe
II
. These results indicate that in the COHb photoproduct the Fe
II
is already high-spin but is closer to the heme plane than in deoxy Hb. Photodissociation from a quintet ligand-field excited state of COHb is suggested. The frequency shifts relative to deoxy Hb persist when the laser pulses are lengthened to 20 ns. The apparently slow relaxation to the fully out-of-plane heme conformation of deoxy Hb is suggested to be associated with change of the globin tertiary structure.
Lens transglutaminase and cataract formation.
Authors: Lorand L , Hsu L K , Siefring G E , Rafferty N S
Published: May 2006

A protein polymer characteristically present in human cataract was shown to contain significant amounts of gamma-glutamyl-epsilon-lysine isopeptides. It is proposed that these crosslinks are produced by the action of transglutaminase (R-glutaminyl-peptide:amine-gamma-glutamyl-yltransferase, EC 2.3.2.13), which is all the more plausible because lens contains the enzyme and endogenous protein substrates for it. The enzyme is similar to that obtained from liver and is Ca2+ dependent. Highest apparent activity is found in lens cortex. When cortex homogenate from the rabbit was incubated in the presence of Ca2+ with either [14C]putrescine or with dansylcadaverine, a a selective incorporation of the radioactive or fluorescent amine into the heavier subunits (Mr approximately 26,000 and 30,000) of beta-crystallins could be demonstrated. Possible modes of regulating the crosslinking activity of this enzyme in lens are discussed.
Mapping a eukaryotic promoter: a DNA sequence required for in vivo expression of adenovirus pre-early functions.
Authors: Osborne T F , Schell R E , Burch-Jaffe E , Berget S J , Berk A J
Published: May 2006

This study defines a DNA sequence upstream from the mRNA cap site required for in vivo expression of the adenovirus 2 pre-early region. The adenovirus 2 pre-early region and flanking sequences were cloned in Escherichia coli plasmid pBR322. Derivatives of the plasmid lacking portions of the upstream viral DNA sequence were constructed. An assay was devised to test the ability of these plasmid DNAs to complement an adenovirus 5 mutant with a deletion in the pre-early region. Plasmids that retained at least 38 base pairs upstream from the mRNA cap site had complementing activity similar to that of the original plasmid, which contains 229 base pairs of upstream viral sequence. However, plasmids retaining 23 or fewer base pairs of viral sequence upstream from the cap site had significantly reduced complementing activity. These results indicate that a portion of the adenovirus 2 sequence between 23 and 38 base pairs upstream from the mRNA cap site is required for expression of the pre-early region. This interval includes the Goldberg-Hogness box-like sequence T-A-T-T-T-A-T-A.
Contacts between DNA gyrase and its binding site on DNA: features of symmetry and asymmetry revealed by protection from nucleases.
Authors: Morrison A , Cozzarelli N R
Published: May 2006

DNA gyrase supercoils DNA by passing one DNA segment through another by means of a reversible double-strand break at specific DNA sites. We determined the nucleotide sequence of two highly preferred gyrase binding sites and analyzed the grip of gyrase on the DNA by using protection from nuclease attack. The DNA-breakage site of gyrase was centered in about 50 base pairs (bp) of DNA that was completely protected from DNase I and flanked by DNA regions cut at average intervals of 9.9 bases. The same pattern of protection from DNase I was observed with topoisomerase II', an enzyme that shares structural homology with gyrase. The gyrase site of DNA breakage was off-center in the 140 bp of DNA protected from exonuclease III digestion. ATP or inhibitors of gyrase had little specific effect on DNase I protection. On addition of a nonhydrolyzable analogue of ATP, previously stable barriers to exonuclease III were invaded and new barriers appeared. We discuss a detailed model uniting these results with previous data on gyrase structure and mechanism.
Cloning and expression of the Pst I restriction-modification system in Escherichia coli.
Authors: Walder R Y , Hartley J L , Donelson J E , Walder J A
Published: May 2006

Here we report the cloning and preliminary characterization of the Pst I restriction-modification system of Providencia stuartii 164. Transformants of Escherichia coli carrying the Pst I gene system inserted into the cloning vector pBR322 were selected on the basis of acquired resistance to bacteriophage lambda infection. Pst I endonuclease was detected in osmotic shock fluid from each of the resistant clones. Plasmid and chromosomal DNA from these clones could not be digested by Pst I, indicating that the gene for the corresponding modification enzyme had also been cloned and was being expressed. The smallest recombinant plasmid encoding both activities, pPst201, contains an insert of approximately 4000 base pairs. In vitro transcription studies indicate that this DNA fragment also contains the endogenous promoter(s) of the system. When pPst201 was introduced into a minicell-producing strain of E. coli, two new proteins, 32,000 and 35,000 daltons, were synthesized. We have assigned these to the Pst I modification (methylase) and restriction enzymes, respectively. The active form of the restriction enzyme is a dimer, as determined by gel filtration. Constructed transformants of P. stuartii 164 that carry the Pst I system inserted into pBR322 produce approximately 10 times more Pst I endonuclease activity than does the native strain.
Formation of 11-trans slow reacting substances.
Authors: Atrache V , Sok D E , Pai J K , Sih C J
Published: May 2006

Under strongly basic conditions [excess LiOH, dimethoxyethane/water (4:1, vol/vol)], purified slow reacting substances (SRSs) SRS-GSH and SRS-Cys were not isomerized to their corresponding 11-trans isomers. However, addition of thiols such as glutathione (GSH) or L-cysteine to this basic medium produced various amounts of 11-trans-SRS, depending on the thiol concentration. This chemical isomerization was inhibited by the radical scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidinooxy free radical (HTMP); the inhibition suggests that the thiyl radical (RS) is added reversibly to the triene system at C-12, resulting in the overall cis leads to trans isomerization of the 11,12 double bond. Because the amount of 11-trans-SRS-Cys produced by intact rat basophilic leukemia (RBL-1) cells was consistently higher than the amount produced in boiled cells, we believe that intact RBL-1 cells contain enzyme systems that form peroxides, which are known to enhance the formation of thiyl radicals, required for cis leads to trans isomerization. Likewise, HTMP inhibited the formation of 11-trans-SRS-Cys in this cell system.
Aggregation of colloidal particles modeled as a dynamical process.
Authors: Bentz J , Nir S
Published: May 2006

Aggregation kinetics of sonicated phosphatidylserine (PtdSer) vesicles in NaCl indicate that the process is fully reversible and dynamical, involving the rapid formation and dispersal of aggregates. Accordingly, the general mass action kinetic equations are analyzed with respect to the equilibrium state and the formation of higher order aggregates. For a general class of systems, the values for the mass average aggregate size at equilibrium are obtained from simple closed-form expressions. It is shown that an analysis of the aggregation equilibrium will yield estimates for the potential energy well that holds the aggregates together. A fit to the experimental data for kinetics of Na+-induced aggregation of the vesicles has been achieved by employing mass action kinetic equations that include the dissociation reactions. The threshold of NaCl concentration required for aggregation involves the clear distinction between the rate and extent of aggregation.
Acoustic microscopy of living cells.
Authors: Hildebrand J A , Rugar D , Johnston R N , Quate C F
Published: May 2006

This paper reports preliminary results of the observation by acoustic microscopy of living cells in vitro. The scanning acoustic microscope uses high-frequency sound waves to produce images with submicrometer resolution. The contrast observed in acoustic micrographs of living cells depends on the acoustic properties (i.e., density, stiffness, and attenuation) and on the topographic contour of the cell. Variation in distance separating the acoustic lens and the viewed cell also has a profound effect on the image. When the substratum is located at the focal plane, thick regions of the cell show a darkening that can be related to cellular acoustic attenuation (a function of cytoplasmic viscosity). When the top of the cell is placed near the focal plane, concentric bright and dark rings appear in the image. The location of the rings can be related to cell topography, and the ring contrast can be correlated to the stiffness and density of the cell. In addition, the character of the images of single cells varies dramatically when the substratum upon which they are grown is changed to a different material. By careful selection of the substratum, the information content of the acoustic images can be increased. Our analysis of acoustic images of actively motile cells indicates that leading lamella are less dense or stiff than the quiescent trailing processes of the cells.
Relaxation processes for a three-wave interaction model
Authors: Meiss James D. , Watson Kenneth M.
Published: May 2006

A system of waves coupled by three-wave, or triad, interactions is considered. A model is constructed to describe the response of a single test wave to interaction with a “heat bath” of “ambient waves.” An exact solution for the relaxation of the test wave amplitude is obtained. Regimes of validity are shown for the use of a Langevin equation based on a two-time scale perturbation theory.
Covalent crosslinking of human chorionic gonadotropin to its receptor in rat testes.
Authors: Rebois R V , Omedeo-Sale F , Brady R O , Fishman P H
Published: May 2006

The bifunctional crosslinking reagents disuccinimidyl suberate and dithiobis(succinimidyl propionate) were used to attach 125I-labeled human chorionic gonadotropin (125I-hCG) covalently to rat testicular membranes. The extent of crosslinking was dependent on time and concentration; routinely, 30% of the specifically bound hormone was covalently linked to the membranes in the presence of 0.5 mM crosslinking reagent when incubated at 25 degrees C for 15 min. Excess unlabeled hCG blocked the crosslinking of 125I-hCG to the membranes. When solubilized with Triton X-100 and analyzed by sucrose density gradient centrifugation, both the native and the crosslinked hormone-receptor complex sedimented with an apparent Mr of 220,000. Thus, the receptor itself would have Mr 180,000. When the crosslinked complex was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the predominant species had a Mr of 123,000 and appeared to represent the labeled alpha subunit of hCG covalently linked to a membrane component. The Mr of this receptor component would be 100,000, a value approximately half that of the Triton X-100-solubilized receptor. Thus, the membrane receptor for hCG may consist of a dimer of two binding subunits or a binding subunit associated with one or more additional subunits that might play a coupling or regulatory function.
Translation of phenylalanine hydroxylase-specific mRNA in vitro: evidence for pretranslational control by glucocorticoids.
Authors: Chiappelli F , Haggerty D F , Lynch M , Popják G
Published: May 2006

We have found that the induction of phenylalanine hydroxylase by hydrocortisone and serum in confluent cultures of H4-II-E-C3 rat hepatoma cells is accompanied by an increase in polysomal mRNA specific for phenylalanine hydroxylase, as measured by translation in a cell-free protein-synthesizing system. Thus, the induction is mediated largely, if not entirely, by a pretranslational mechanism, possibly by stimulation of the transcription of the phenylalanine hydroxylase gene.
beta-Endorphin: characteristics of binding sites in a neuroblastoma--glioma hybrid cell.
Authors: Hammonds R G , Ferrara P , Li C H
Published: May 2006

Specific binding of human beta-endorphin to NG108-15 cells is described; human beta-[Tyr27-3H2] endorphin was used as the ligand. The binding is time dependent and saturable; Kd = 0.3 nM and ka = 1.8 x 10(8) M-1 min-1. Under the conditions optimal for beta-endorphin binding, leucine-enkephalin has one-fourth to one-third as many binding sites as beta-endorphin and its affinity is 7--10% that of beta-endorphin. Monovalent and divalent cations potently inhibit binding. Trypsin, phospholipase A, and N-ethylmaleimide reduce the ability of NG108-15 cells to bind beta-endorphin. beta-Endorphin analogs are able to fully inhibit the binding of beta-[Tyr27-3H2]endorphin, although enkephalins, morphine, and naloxone inhibit only 50--80%.
Inhibition of vaccinia mRNA methylation by 2',5'-linked oligo(adenylic acid) triphosphate.
Authors: Sharma O K , Goswami B B
Published: May 2006

Extracts of interferon-treated cells synthesize unique 2',5'-linked oligo(adenylic acid) 5'-phosphates in the presence of ATP and double-stranded RNA. 2',5'-linked oligo(adenylic acid) 5'-triphosphate inhibits protein synthesis at nanomolar concentrations by activating RNase. We have observed that oligo(adenylic acid) 5'-monophosphate and 5'-triphosphate are potent inhibitors of vaccinia mRNA methylation in vitro. Both the methylation of mRNA methylation is not due to degradation of the mRNA. Inhibition of the 5'-terminal guanine at the 7 position and the 2'-O-ribose methylation of the penultimate nucleoside are inhibited. Such inhibition of the requisite modification of the 5' terminus of mRNA by 2',5'-linked oligo(adenylic acids) may be a mechanism of interferon action against both DNA and RNA viruses in which mRNAs derived from them are capped.
Mouse beta-globin and adenovirus-2 major late transcripts are initiated at the cap site in vitro.
Authors: Hagenbüchle O , Schibler U
Published: May 2006

Initiation of transcription on major late DNA of adenovirus serotype 2 and the beta-globin gene of the mouse was studied by using ribonucleoside [beta-32P]triphosphates as labeled precursors in vitro. The 5' termini of discrete runoff transcripts of both templates were preferentially labeled. The beta phosphate of the cap structure was labeled by [beta-32P]ATP but not [beta-32P]GTP, indicating that the beta phosphate of the cap is contributed by the initiating nucleoside triphosphate (ATP in both cases). Therefore, capping occurs on the initial nucleotide of these transcripts.
Host restriction of Friend leukemia virus: gag proteins of host range variants.
Authors: Duttagupta S , Soeiro R
Published: May 2006

The host response to murine ecotropic leukemia viruses is mainly controlled by the mouse Fv-1 gene. This locus controls virus replication at an intracellular stage and prevents provirus integration. Biological studies suggest that the Fv-1 effector molecule recognizes at least one virion structural protein. We have produced host range variants of B-tropic Friend murine leukemia virus in order to study the primary structure of potential viral target proteins. Our results show that conversion of B-tropism to NB-tropism is associated with changes in the primary structure of three gag proteins--p15, p12, and p30. These results suggest that host range conversion is due to a recombinational event, presumably between the parental virus and an endogenous murine virus. They also open the possibility that p12 and p30 may be involved in host range restriction.
Increased mitochondrial uptake of rhodamine 123 during lymphocyte stimulation.
Authors: Darzynkiewicz Z , Staiano-Coico L , Melamed M R
Published: May 2006

The positively charged rhodamine analog rhodamine 123 accumulates specifically in the mitochondria of living cells. In the present work, the uptake of rhodamine 123 by individual lymphocytes undergoing blastogenic transformation in cultures stimulated by phytohemagglutinin was measured by flow cytometry. A severalfold increase in cell ability to accumulate rhodamine 123 was observed during lymphocyte stimulation. Maximal dye uptake, seen on the third day of cell stimulation, coincided in time with the peak of DNA synthesis (maximal number of cells in the S phase) and mitotic activity. A large intercellular variation among stimulated lymphocytes, with some cells having fluorescence increased as much as 15 times in comparison with nonstimulated lymphocytes, was observed. Whereas the increased uptake of rhodamine 123 also correlated with the increase in cellular RNA content, the correlation between the dye uptake and cell size (measured by light scatter) was less apparent. As observed by UV microscopy, the increased dye uptake during the blastogenesis was due, to a large extent, to an increase in number of mitochondria per cell. However, an additional increase in rhodamine 123 binding per mitochondrion or per unit of mitochondrial membrane in stimulated cells could not be excluded. The present data indicate that rhodamine 123 may be used as a supravital mitochondrial probe, discriminating between cycling and quiescent cells and having application in sorting functionally distinct cell subpopulations.
Mechanism of photosensitivity in systemic lupus erythematosus patients.
Authors: Emerit I , Michelson A M
Published: May 2006

Patients who have systemic lupus erythematosus have increased numbers of chromosome breaks and rearrangements correlated with a low molecular weight chromosome-damaging agent that is released from their lymphocytes into the serum. This clastogenic factor also produces chromosome breaks and sister chromatid exchanges in healthy persons' lymphocytes when they are incubated in the presence of lupus patients' serum or lymphocytes or purified factor. The lymphocytes from lupus patients are sensitive to near-UV (360- to 400-nm light. This sensitivity seems to be related to the presence of the clastogenic factor in these cells; lymphocytes of healthy persons exposed to the factor also become sensitive to light of the same wavelengths. A significant increase in nonviable cells (trypan blue exclusion test) was observed after 5 min of irradiation with 360- and 380-nm light in the presence of the factor. The number of chromosome aberrations observed after stimulation of the irradiated lymphocytes with phytohemagglutinin was also maximal after irradiation at 380 nm in presence of the factor. The combined action of near-UV light plus clastogenic factor was inhibited by superoxide dismutase if the enzyme were present during irradiation, suggesting that activation involves photoproduction of superoxide ions. Irradiation of the purified factor and immediate addition of it to lymphocytes gave the same results whereas preirradiation of cells or of medium was without effect. The presence of this photoactivated agent explains why patients who have lupus erythematosus show an aggravated condition after exposure to sunlight and the appearance of typical skin lesions.
Defects of receptor-mediated low density lipoprotein catabolism in homozygous familial hypercholesterolemia and hypothyroidism in vivo.
Authors: Thompson G R , Soutar A K , Spengel F A , Jadhav A , Gavigan S J , Myant N B
Published: May 2006

The role of low density lipoprotein (LDL) receptors in the pathogenesis of hereditary and acquired forms of hypercholesterolemia has been investigated in vivo by simultaneously determining total and receptor-independent LDL catabolism with 125I-labeled LDL and 131I-labeled LDL coupled with cyclohexanedione. Receptor-mediated catabolism of LDL, determined as the difference between the turnover of 125I and 131I, was found to be virtually absent in two homozygotes with familial hypercholesterolemia and markedly reduced in a hypothyroid patient. Treatment of the latter with L-thyroxine markedly stimulated receptor-mediated catabolism and reduced LDL levels as did cholestyramine administration in a control subject. Reduction of LDL levels by plasma exchange in a control subject and homozygote had no such effect. These results demonstrate the existence of an intrinsic and almost total defect of receptor-mediated LDL catabolism in homozygous familial hypercholesterolemia and demontrate an analogous but reversible abnormality in hypothyroidism.
Specific binding of a prokaryotic ribosomal protein to a eukaryotic ribosomal RNA: implications for evolution and autoregulation.
Authors: Gourse R L , Thurlow D L , Gerbi S A , Zimmermann R A
Published: May 2006

Ribosomal protein L1 from the prokaryote Escherichia coli has been shown to form a specific complex with 26S ribosomal RNA from the eukaryote Dictyostelium discoideum. The segment of Dictyostelium rRNA protected from ribonuclease digestion by L1 and the corresponding region in Dictyostelium rDNA were investigated by nucleotide sequence analysis, and an analogous section in rDNA from Xenopus laevis was identified. When the L1-specific segments from eukaryotic rRNA were compared with those from prokaryotic rRNA, striking similarities in both primary and secondary structure were apparent. These conserved features suggest a common structural basis for protein recognition and indicate that such regions became fixed at a very early stage in rRNA evolution. In addition, certain structural elements of the L1 binding sites in rRNA are also found in the initial segment of the polycistronic L11-L1 mRNA, providing support for the hypothesis that L1 participates in the regulation of ribosomal protein synthesis by specific interaction with its own mRNA.
Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli.
Authors: Schnepf H E , Whiteley H R
Published: May 2006

Sau 3A1 partial digestion fragments from Bacillus thuringiensis var. kurstaki HD-1 plasmid DNA were ligated into the BamHI site of the cloning vector pBR322 and transformed into Escherichia coli strain HB101. Colonies presumed to contain recombinant plasmids were screened for production of an antigen that would react with antibody made against B. thuringiensis crystals. One strain, ES12, was isolated by using this procedure. ES12 contains a plasmid of Mr 11 X 10(6) that has DNA sequence homology with pBR322 as well as with Mr 30 X 10(6) and Mr 47 X 10(6) plasmids of B. thuringiensis. It makes a protein antigen, detected by antibodies to crystal, which has the same electrophoretic mobility as the B. thuringiensis crystal protein. Protein extracts of ES12 are toxic to larvae of the tobacco hornworm Manduca sexta.
The polyribosomal mRNA--protein complex is a dynamic structure.
Authors: Greenberg J R
Published: May 2006

The metabolism of mRNA--protein complexes present in polyribosomes of mouse L cells was investigated with the aid of ultraviolet light-induced crosslinking of proteins to RNA. It was shown that a set of at least seven proteins which are synthesized and become associated with mRNA under conditions permitting mRNA synthesis also are synthesized and become associated with mRNA when mRNA synthesis is inhibited with a high dose of actinomycin D. This set includes a protein of Mr 78,000 which can be crosslinked to poly(A). These findings imply that mRNA-associated proteins exchange in the cytoplasm with a pool of free proteins. Taken together with results from other laboratories, they suggest a role for mRNA-associated proteins in translation. The labeling kinetics of mRNA-associated proteins were also investigated and found to be consistent with cytoplasmic addition to mRNA and a half-life of more than 2 hr. Additional implications of these findings are discussed.
Mutations that affect lamB gene expression at a posttranscriptional level.
Authors: Schwartz M , Roa M , Débarbouillé M
Published: May 2006

We previously obtained strains of Escherichia coli in which the beginning of gene lacZ, which codes for beta-galactosidase, is replaced by the beginning of gene lamB, which codes for a maltose-inducible outer membrane protein. In some of these strains the induction (with maltose) of lamB-lacZ hybrid protein synthesis was lethal because of membrane damage resulting from an incomplete export of this protein to the outer membrane. We describe here a class of maltose-resistant mutants obtained from one such strain. Mutants in this class fail to produce the lamB-lacZ hybrid protein but retain the ability to express lacY, which is located distal to the hybrid gene. Some of the mutants carry deletions within the hybrid gene. The others carry point mutations which most probably affect the initiation of translation at the beginning of the hybrid gene. One of these is located in the sequence that codes for the presumed ribosome interaction site on the mRNA. Three others, of which two are located in the coding region (sixth codon), are believed to result in an alteration of mRNA secondary structure such that the accessibility of the ribosome interaction site is reduced.
Human embryonic somatomedin.
Authors: Sara V R , Hall K , Rodeck C H , Wetterberg L
Published: May 2006

A radioreceptor assay utilizing human fetal brain plasma membrane as matrix and somatomedin A as ligand (fetal brain RRA-SMA) was developed. Increased levels of fetal brain RRA-SMA were found in the fetal circulation. The concentration was approximately 4-fold higher in the fetal as compared to the adult human. At birth, values fell within the adult range. In contrast, adult somatomedins determined by somatomedin radioimmunoassay were undetectable in the fetus and below the adult range at birth. Levels of fetal brain RRA-SMA were decreased in fetuses with different clinical disorders. In healthy newborns at cesarean section a significant correlation between serum fetal brain RRA-SMA values and birth weight and length was found. These results indicate the presence of an embryonic somatomedin in humans. The fetal brain RRA-SMA may provide a reliable index of fetal growth.