bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo
induction of normal differentiation in myeloid leukemia cells
Authors: Lotem Joseph , Sachs Leo
Published: May 2006

MGI
+
D
+
, MGI
+
D
-
, and MGI
-
D
-
mouse myeloid leukemic cells, which genetically differ in their competence to be induced to undergo normal cell differentiation
in vitro
by the normal macrophage- and granulocyte-inducing protein MGI, were analyzed for their ability to undergo cell differentiation in diffusion chambers
in vivo
. As after induction by MGI
in vitro
, MGI
+
D
+
clones were induced for Fc and C3 rosettes, lysozyme, and mature macrophages and granulocytes in normal syngeneic or allogeneic mice. MGI
+
D
-
clones were also induced in these mice for all these properties, although
in vitro
they were not induced by MGI for mature cells. The MGI
-
D
-
clones were induced
in vivo
for C3 and Fc rosettes, lysozyme, and intermediate stages but not for mature cells, whereas none of these properties were induced in these clones by MGI
in vitro
. Thus, certain types of myeloid leukemic cells differentiate better
in vivo
, possibly due to the presence of higher effective concentrations of MGI and/or other inducing factors, and MGI
+
D
+
and MGI
+
D
-
cells can completely differentiate
in vivo
to mature cells.
In vivo
differentiation was inhibited in mice treated with cyclophosphamide. It was also inhibited in various strains of
nude
mice, except for one MGI
+
D
+
clone, where it was inhibited in C57BL/6 but not in ICR
nude
mice. This MGI
+
D
+
clone was also the only clone that was induced to differentiate normally
in vitro
by a 23,000 molecular weight form of purified MGI. The results suggest that different clones respond to different molecular forms of MGI, which may be present in different proportions in some animals, that
in vivo
differentiation by MGI possibly with other factors may be regulated by cells involved in the immune response, and that this differentiation can be genetically controlled. Differentiation
in vivo
was enhanced by injection of conditioned medium containing MGI and by inoculation of MGI-producing cells, including normal granulocytes. This indicates that the induction of normal differentiation of myeloid leukemic cells
in vivo
can be enhanced by these treatments.
Growth factors from murine sarcoma virus-transformed cells.
Authors: de Larco J E , Todaro G J
Published: May 2006

Murine sarcoma virus-transformed mouse fibroblasts produce polypeptide growth factors and release them into serum-free medium. These factors stimulate cells to divide in monolayer cultures and also to form colonies that grow progressively soft agar. Three major peaks of activity are seen, with apparent molecular weights of 25,000, 12,000, and 7000. The sarcoma growth factors are heat-stable, trypsin-sensitive, and active in nanogram quantities when tested for growth stimulation of untransformed rat and mouse fibroblasts. All three molecular species are also capable of competing for membrane epidermal growth factor (EGF) receptors when tested with 125I-labeled EGF. They differ from mouse EGF, however, in their molecular weights, in their inability to react with anti-EGF antibodies, and in their ability to convert cells to anchorage independent (agar) growth. For the above reasons, we conclude that the sarcoma growth factors are a new class of polypeptide tropic factors that confer on fibroblasts in vitro properties associated with the transformed phenotype.
Steering responses of flying crickets to sound and ultrasound: Mate attraction and predator avoidance
Authors: Moiseff Andrew , Pollack Gerald S. , Hoy Ronald R.
Published: May 2006

We investigated the steering responses of tethered, flying adult female crickets (
Teleogryllus oceanicus
) to acoustic stimulation. Crickets responded directionally to directional sound stimulation by bending their abdomens and hind legs to one side. We interpret this response as an attempt to turn. When stimulated with a model of conspecific calling song with a carrier frequency of from 3 to 9 kHz, crickets turned toward the sound source. We believe that this indicates a positive phonotactic response of flying females to calling, conspecific males. When offered a choice between conspecific song and the song of another species, females turned exclusively toward conspecific song, demonstrating that the response is species specific. The direction of the response is dependent on the carrier frequency of the song, and it demonstrates frequency discrimination. Females turned toward calling song when it was played at carrier frequencies from 3 to 9 kHz, but they turned away from the same song pattern played at carrier frequencies from 30 to 70 kHz. This negative phonotaxis to ultrasonic stimuli suggests that crickets, like some other nocturnal flying insects, can evade bats by acoustic detection.
Antagonists of DNA gyrase inhibit repair and recombination of UV-irradiated phage lambda.
Authors: Hays J B , Boehmer S
Published: May 2006

Intracellular lambda DNA (from EDTA-sensitive tandem duplication phages) was extracted from infected rec+ bacteria and scored for infectivity and recombination (loss of duplication) by transfection of recA recB spheroplasts and subsequent assay for EDTA resistance. When phage development was blocked by repressor or by antibiotics (chloramphenicol and/or rifampin), the apparent recombination frequency was about 0.1% above the background value for recA infections. Prior irradiation of the phage greatly stimulated recombination; the frequency was 20% when UV fluence was 140 J/m2. Repair (recovery of infectivity) and recombination of irradiated phage DNA proceeded readily in the presence of chloramphenicol and rifampin. Inhibitors of DNA gyrase (coumermycin and oxolinic acid) blocked repair and reduced recombination. UV-stimulated recombination was very low in recA but nearly normal in recB cells: repair was reduced in both mutant strains. The recombination remained high as phage/cell ratios less than unity.
3'-terminal nucleotide sequence of encephalomyocarditis virus RNA determined by reverse transcriptase and chain-terminating inhibitors.
Authors: Zimmern D , Kaesberg P
Published: May 2006

We have adapted the chain-termination method for determining the nucleotide sequence of DNA of Sanger, Nicklen, and Coulson [(1977) Proc. Natl. Acad. Sci. USA 74, 5463--5467] for use with reverse transcriptase (RNA-directed DNA nucleotidyltransferase) on RNA templates. With this method and using a primer (the octanucleotide pdT7rC) directed at the 3'-terminal poly(A), we have determined a sequence of 166 residues in the genomic RNA of the picornavirus encephalomyocarditis virus.
Rate of macromolecular synthesis through the cell cycle of the yeast Saccharomyces cerevisiae.
Authors: Elliott S G , McLaughlin C S
Published: May 2006

Centrifugal elutriation was used to separate cells of Saccharomyces cerevisiae in balanced exponential growth according to position in the cell cycle. Macromolecular synthesis was examined. DNA synthesis was found to be periodic, but RNA and protein synthesis showed an exponential increase in rate. Two-dimensional electrophoresis was used to determine the rate of synthesis of individual proteins, with 111 of the more abundant cellular proteins selected for analysis from among the more than 1000 proteins that migrate in the system. All the examined proteins showed an exponentially increasing rate of synthesis.
2,5-Dimethylfuran from ≈2.7 × 10
9
-year-old Rupemba—Belingwe stromatolite, Rhodesia: Potential evidence for remnants of carbohydrates
Authors: Sklarew Deborah S. , Nagy Bartholomew
Published: May 2006

2,5-Dimethylfuran, 2-methylfuran, and C
9
—C
20
n
-alkanes have been isolated from the kerogen of a Rupemba stromatolite. These furans could be the oldest known stable remnants of biological carbohydrates if it is possible to prove that their progenitors are as old as the rock. Experiments showed that it is improbable that these compounds are analytical artifacts or laboratory contaminations. Postdepositional contaminations caused by fluids flowing through the rocks seem doubtful because of minimal permeability, which is supported by petrographic observations. Contamination by diffusion also appears to be unlikely. Because the rock had a mild temperature history, these molecules could have survived since Archean time. Because of all the evidence, the furans and alkanes are quite likely biochemical fossils.
Formation and properties of 1000-A-diameter, single-bilayer phospholipid vesicles.
Authors: Enoch H G , Strittmatter P
Published: May 2006

Two methods are reported for the formation of large, uniform-sized phospholipid vesicles. The methods involve the treatment of phospholipid, in the form of either small, sonicated vesicles or a dry lipid film, at a molar ratio of deoxycholate to phospholipid of 1:2. Subsequent removal of deoxycholate yields a stable preparation of vesicles. These vesicles are bounded by a single bilayer, have an average diameter of 1000 A, and are readily separated from sonicated vesicles (230 A) by gel filtration on Sepharose 4B. Since the 1000-A vesicles are capable of trapping enzymes and other macromolecules, they may prove valuable for the delivery of liposome-entrapped solutes to cells and for the localization of peptide segments of a spectrum of membrane-bound proteins.
Sterigmatocystin-DNA interactions: identification of a major adduct formed after metabolic activation in vitro.
Authors: Essigmann J M , Barker L J , Fowler K W , Francisco M A , Reinhold V N , Wogan G N
Published: May 2006

Sterigmatocystin (ST), a potent hepatocarcinogen, was covalently bound to calf thymus DNA by incubation in the presence of phenobarbital-induced rat liver microsomes. Acid hydrolysis of ST-modified DNA liberated a major guanine-containing adduct, present in DNA at an estimated level of 1 ST residue per 100-150 nucleotides. The adduct was isolated by high-pressure liquid chromatography and subjected to structural analysis. Spectral and chemical data identified the adduct as 1,2-dihydro-2-(N(7)-guanyl)-1-hydroxysterigmatocystin, the guanine and hydroxyl moieties being in a trans configuration. The structure and stereochemistry of this adduct indicated that the exo-ST-1,2-oxide was the metabolite that reacted with DNA, and the quantitative yield of adduct indicated that this metabolite was a major product of the in vitro metabolism of ST.
Calcium-sensitive regulation of actin-myosin interactions in baby hamster kidney (BHK-21) cells.
Authors: Yerna M J , Dabrowska R , Hartshorne D J , Goldman R D
Published: May 2006

A fraction has been obtained from baby hamster kidney (BHK-21) cells that will stimulate the actin-moderated ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of both BHK-21 myosin and gizzard smooth muscle myosin. This activation is associated with the specific phosphorylation of the myosin 20,000-dalton light chain. The BHK-21 myosin light chain kinase preparation contains a major protein of approximately 105,000 molecular weight as determined by sodium dodecyl sulfate gel electrophoresis. Both the actin activation and phosphorylation events require the presence of Ca2+ and the so-called modulator or calcium-dependent regulator protein that has been isolated from smooth muscle, brain, and other tissues. On the basis of these results we propose that this kinase system constitutes a Ca2+-dependent regulatory mechanism for myosin-actin interactions in nonmuscle mammalian cells.
Conformational properties of the acetylcholine receptor as revealed by studies with constrained depolarizing ligands
Authors: Wassermann N. H. , Bartels E. , Erlanger B. F.
Published: May 2006

Conformational aspects of the acetylcholine receptor (AcChoR) of
Electrophorus electricus
have been examined by studies of its interaction with structurally related, constrained aromatic bis quaternary compounds. Among the compounds synthesized was 3,3′-bis[α-(trimethylammonium)-methyl]azobenzene dibromide (3,3′-bisQ). This compound is photochromic and can exist in a
cis
or
trans
isomeric form, both of which have now been isolated in pure form.
Trans
-3,3′-bisQ is the most potent activator known, producing a 60-mV depolarization at 0.2 μM and 50% activity at 0.06 μM. The
cis
isomer is less than 1% as active. Its high activity and constrained structure suggest that
trans
-3,3′-bisQ can be considered to be a “template” of the combining site of AcChoR, when the latter is in the activated state. The following conclusions can then be drawn concerning the AcChoR binding site. (
i
) Depolarization can occur by interaction with reagents that are essentially inflexible. (
ii
) The binding site has a planar hydrophobic region that interacts with methylene groups of acetylcholine and with hydrophobic areas in general. (
iii
) In the same plane as the hydrophobic area is a site that interacts with electron-donating functional groups including the carbonyl oxygen of acetylcholine and the azo nitrogens of
trans
-3,3′-bisQ. (
iv
) About 1.5 Å out of the plane of the hydrophobic and the electron acceptor site is an anionic site; when the AcChoR is in the activated state, this site is separated from the electron acceptor site by 5.2 Å and from another anionic site by 11 Å. (
v
) The anionic sites are located within a cleft of limited size, sufficient to accommodate quaternary methyl groups. (
vi
) Although depolarization can occur with reagents that possess only hydrophobic and cationic groups if their geometric arrangement is proper, the highest activity resides in compounds capable of all of the interactions cited above.
Relationship between RNA content and progression of lymphocytes through S phase of cell cycle.
Authors: Darzynkiewicz Z , Evenson D , Staiano-Coico L , Sharpless T , Melamed M R
Published: May 2006

A flow cytometric technique for simultaneous measurements of RNA and DNA in individual cells has been applied to correlate the content of cellular RNA with the rate of progression of cells through the S phase. Human peripheral lymphocytes stimulated with phytohemagglutinin were blocked at the G1/S phase boundary by hydroxyurea or 5-fluorodeoxy-uridine treatment. Cells in the G1 phase as well as cells blocked at the G1/S phase boundary showed high heterogeneity with respect to stainable RNA content. After release from the block, the cells traversed the S phase at rates proportional to the quantity of stainable RNA per cell. Cells with the highest RNA content completed DNA replication 5 hr after release from the block; the cells with minimal RNA traversed the S phase at one-fifth of this rate. The large intercellular variation in stainable RNA and length of the S phase may be due to functional heterogeneity in the lymphocyte population. Our results suggest a correlation between the number of ribosomes and the rate of DNA replication in lymphocytes.
Strongly nonlinear parabolic initial-boundary value problems
Authors: Brezis Haim , Browder Felix E.
Published: May 2006

An existence and uniqueness result is presented for the solution of a parabolic initial-boundary value problem under Dirichlet null boundary conditions for a general parabolic equation of order 2
m
with a strongly nonlinear zeroth-order perturbation. This is the parabolic generalization of a class of elliptic results considered earlier by the writers and others and is based upon a new compactness theorem.
Genetic translocation in Staphylococcus aureus.
Authors: Novick R P , Edelman I , Schwesinger M D , Gruss A D , Swanson E C , Pattee P A
Published: May 2006

A 5.2-kilobase pair transposon, Tn551, has been found in Staphylococcus aureus, a Gram-positive bacterium. Initially detected on plasmid pI258, it undergoes rec-independent transposition to multiple chromosomal and plasmid sites, sometimes causing insertional inactivation. Unlike most other transposons, Tn551 undergoes apparently precise excision as a rule. The initial observation of Tn551 transition involved UV inactivation of the carrier plasmid; this would appear to be a general means of detecting transposable elements.
Photomutagenesis by chlorinated phenothiazine tranquilizers.
Authors: Jose J G
Published: May 2006

Phenothiazine tranquilizers are widely used pharmaceuticals that have been associated with side effects, such as formation of cataracts, that seem related to light exposure. Because patients may use them over extensive time periods, it is important to determine what deleterious cellular effects these drugs may cause and, if possible, to select or design drugs that do not cause such effects. The results reported here demonstrate that chlorinated phenothiazine drugs can be photoactivated to mutagenic species, whereas the nonchlorinated analogues do not possess this characteristic. None of the phenothiazines tested is mutagenic in the dark. Mutagenicity was observed only in strains of Salmonella typhimurium that lacked excision repair of DNA, and the mutagenicity was elevated in strains that contained the plasmid pKM101, which may enhance error-prone repair.
Principles of polymorphism and epistasis for multilocus systems
Authors: Karlin Samuel
Published: May 2006

The nature of stable equilibrium configurations is described for general nonepistatic and generalized symmetric viability regimes in multilocus systems under conditions of tight and loose linkage. The influence of epistasis and symmetry can be better understood in terms of these standards. A dichotomy in the nature of stable polymorphisms emerges. More recombination, bisexuality, and multideme interactions facilitate the establishment of central type polymorphisms.
Reliable
ab initio
calculation of a chemical reaction rate and a kinetic isotope effect: H + H
2
and
2
H +
2
H
2
Authors: Garrett Bruce C. , Truhlar Donald G.
Published: May 2006

We calculate equilibrium rate constants for
ortho-para
conversion in hydrogen and deuterium by an atomic mechanism. The calculations are based on an accurate
ab initio
potential surface, transition state theory, and an adiabatic transmission coefficient. The calculated rate constants are demonstrated to be reliable within 40-50%, and they agree with experiment within this margin.
Interferon, double-stranded RNA, and RNA degradation: activation of an endonuclease by (2'-5')An.
Authors: Slattery E , Ghosh N , Samanta H , Lengyel P
Published: May 2006

Among the mediators of interferon action are one enzyme that is activated by double-stranded RNA to convert ATP to (2'-5')An and a second enzyme, an endonuclease, that is activated by (2'-5')An to cleave single-stranded RNA. The binding of (2'-5')An to the endonuclease (partially purified from mouse Ehrlich ascites tumor cells) is revealed by its retention on nitrocellulose filters. This can serve as the basis for an assay of the enzyme. Activation of the enzyme is reversible and is lost upon removal of (2'-5')An:gel filtration of activated endonuclease on Sephacryl S-200 results in an inactive enzyme. The enzyme can be activated again, however, by addition of (2'-5')An. The elution volume of the nonactivated endonuclease from Sephadex G-200 indicates that its molecular weight is 185,000, unusually large for a nuclease. The elution volume of the maximally activated endonuclease from Sephadex G-200 equilibrated with (2'-5')An is not detectably different from that of enzyme that had not been previously activated that was passed through Sephadex G-200 not equilibrated with (2'-5')An. This indicates that the activation does not result in a large change in the size or conformation of the enzyme.
Origin of observed changes in
14
N hyperfine interaction accompanying R → T transition in nitrosylhemoglobin
Authors: Mun S. K. , Chang Jane C. , Das T. P.
Published: May 2006

Theoretical investigations of electronic distributions in eight different structural forms of nitrosylhemoglobin were carried out to study the changes in
14
N hyperfine interaction observed with the transition from R to T structures under the influence of inositol hexaphosphate or changing pH. Four of the eight forms studied consisted of protonated and deprotonated N
pros
in the proximal imidazole ligand with linear and bent Fe—N—O structures. Two other forms had a straight Fe—N—O structure and Fe—Im bond stretched by 0.5 and 1.0 Å. The other two systems we have studied are five-liganded NO-heme with bent and straight Fe—N—O structures. Our investigations show that arrangements of energy levels did not differ significantly among all the structures, the unpaired electron always occupying an antibonding orbital with

d
z

2 symmetry. The protonated and deprotonated systems with either linear or bent Fe—N—O structure showed substantial hyperfine interaction of the
14
N nuclei of the NO group and the N
ε
atom of the proximal imidazole, indicating that a 9-line electron spin resonance hyperfine pattern (R structure) would be expected in all four cases. On the other hand, the extensions of the Fe—Im bond produce a sizeable decrease in the
14
N
ε
hyperfine interaction, indicating that an extension beyond 1.0 Å would provide a 3-line hyperfine pattern close to that found for the five-liganded NO-heme system. Our results thus provide quantitative support for the model of severe extension or cleavage of the Fe—N
ε
bond proposed in the literature for explaining the R-to-T transition of the α-chain of nitrosylhemoglobin.
Cell-free synthesis and processing of a putative precursor for mitochondrial carbamyl phosphate synthetase I of rat liver
Authors: Mori Masataka , Miura Satoshi , Tatibana Masamiti , Cohen Philip P.
Published: May 2006

Total RNA or poly(A)
+
RNA of rat liver was translated in a rabbit reticulocyte or wheat germ protein-synthesizing system and the carbamyl phosphate synthetase I [carbamoyl-phosphate synthetase (ammonia); carbon dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16] synthesized was isolated by indirect immunoprecipitation by using antibody purified on enzyme-bound Sepharose and
Staphylococcus aureus
cells. The
in vitro
product moved on sodium dodecyl sulfate/polyacrylamide gels as a polypeptide that was about 5000 daltons larger than the subunit of the mature enzyme (160,000 daltons). The same polypeptide was also obtained by direct immunoprecipitation or by a double-antibody precipitation method. The mature enzyme competed effectively with the
in vitro
product for interaction with anti-carbamyl phosphate synthetase I antibody. Digestion of the
in vitro
product by
S. aureus
protease gave a pattern of peptide fragments similar to that of the mature enzyme. A mitochondrial membrane preparation from rat liver converted the
in vitro
product into a polypeptide that comigrated with the mature subunit on sodium dodecyl sulfate gel electrophoresis. Similar proteolytic activity was not detected in either a cytosol or a microsomal fraction of rat liver. These results indicate that the enzyme is synthesized as a larger precursor which is converted to the mature form of enzyme by posttranslational processing.
A study of the adipose conversion of suspended 3T3 cells by using glycerophosphate dehydrogenase as differentiation marker
Authors: Pairault Jacques , Green Howard
Published: May 2006

The adipose conversion of 3T3 cells has been examined in stabilized suspension cultures. In 3T3-F442A cells, glycerophosphate dehydrogenase (
sn
-glycerol-3-phosphate: NAD
+
2-oxidoreductase, EC 1.1.1.8), a key enzyme in triglyceride synthesis, increases in specific activity by more than 5000-fold and can be used as a sensitive and precise measure of the conversion. The conversion depends on an adipogenic factor present in the serum, and this factor can be assayed by the cellular enzyme response. If the cells are growing at the time they receive the adipogenic factor, the enzyme response does not become detectable until after 3 days, during which the cells first enter a resting state. If the cells are resting at the time they receive the adipogenic factor, the enzyme activity begins to increase in 24 hr or less. Only resting cells seem susceptible to the reprogramming of their differentiated state necessary for the adipose conversion. Once the conversion begins, the increase in enzyme activity is exponential over at least 2 orders of magnitude. When cells in a resting state begin the adipose conversion, their biosynthetic processes are accelerated: the rate of protein synthesis increases, they accumulate cell protein, and they may replicate their DNA and divide. The cell multiplication is not essential for adipose conversion but is a form of clonal selection that increases the proportion of adipose cells relative to nonadipose cells.
Precise localization of human beta-globin gene complex on chromosome 11.
Authors: Gusella J , Varsanyi-Breiner A , Kao F T , Jones C , Puck T T , Keys C , Orkin S , Housman D
Published: May 2006

Cloned DNA probes were used in combination with a panel of five hybrid cell clones containing a series of different terminal deletions in human chromosome 11 to map precisely the human hemoglobin beta and delta chain structural genes contained on this chromosome. The region of deletion in each clone of the panel has been defined by biochemical, immunologic, and cytogenetic markers. DNA from clones containing successively larger terminal deletions was tested with appropriate DNA probes to determine the point on the chromosome at which DNA for these two closely linked hemoglobin genes is deleted. These genes, and by inference the closely linked G gamma and A gamma globin genes as well, have been assigned to the intraband region 11p1205 leads to 11p1208 on the short arm of chromosome 11, an interval containing approximately 4500 kilobases of DNA. The approach appears to have potential for even greater resolution and reasonably wide applicability for gene mapping.
β-Endorphin: Analgesic and hormonal effects in humans
Authors: Foley Kathleen M. , Kourides Ione A. , Inturrisi Charles E. , Kaiko Robert F. , Zaroulis Charles G. , Posner Jerome B. , Houde Raymond W. , Li Choh Hao
Published: May 2006

The pharmacokinetics and the hormonal, analgesic, and behavioral effects of several doses of human β-endorphin were evaluated after intravenous administration to three patients and intracerebroventricular administration to one patient with pain caused by cancer. These effects were compared to the hormonal effects of intravenously administered morphine sulfate in two patients and an enkephalin analog in two baboons. The mean terminal half-life after intravenous administration of 5 or 10 mg of human β-endorphin to three patients was 37 min; the mean volume of distribution was 178 ml/kg, and the metabolic clearance rate was 3.2 (ml/min)/kg. The half-life of β-endorphin in cerebrospinal fluid after intracerebroventricular administration was 93 min, and the volume of distribution was 0.74 ml/kg. A rapid rise in plasma prolactin followed both intravenous and intracerebroventricular β-endorphin. Intravenous administration did not affect plasma growth hormone, but intracerebroventricular administration suppressed plasma growth hormone. No significant change in plasma growth hormone was noted after intravenous administration of morphine to humans, but plasma growth hormone decreased in one baboon after administration of the enkephalin analog. β-Endorphin-stimulated release of prolactin occurred at doses lower than those required to produce analgesic and other behavioral effects. When both hormonal and analgesic effects were observed (after 7.5 mg were given intracerebroventricularly), the onset of the hormonal response slightly preceded the analgesic and behavioral responses. These studies suggest that the hormonal effects of β-endorphin are species dependent and are similar to those of morphine. Hormonal and analgesic effects of β-endorphin appear to result from the activation of opiate receptors that differ in their locations and characteristics.
Attractants and repellents control demethylation of methylated chemotaxis proteins in Escherichia coli.
Authors: Toews M L , Goy M F , Springer M S , Adler J
Published: May 2006

A group of methylated proteins, the methyl-accepting chemotaxis proteins (MCP), has been shown to play a central role in bacterial chemotaxis. Both methylation and demethylation of MCP occur continuously in the absence of added stimuli; these two processes are in balance such that a basal level of methylation is maintained. Attractants cause the methylation level to increase to a new value, whereas repellents bring about a decrease in level. Therefore, attractants and repellents must somehow perturb the balance between methylation and demethylation of MCP. In this report the effect of attractants on demethylation of MCP was monitored in two ways: (i) by following the disappearance of [methyl-3H]MCP and (ii) by measuring formation of [3H]methanol, the product of MCP demethylation. Both methods showed that addition of attractants causes a transient inhibition of MCP demethylation. Repellent addition has previously been shown to stimulate MCP demethylation. It is therefore concluded that control of demethylation plays a crucial role in changing the level of methylation of MCP in response to attractants and repellents.
An endothelial cell growth factor from bovine hypothalamus: identification and partial characterization.
Authors: Maciag T , Cerundolo J , Ilsley S , Kelley P R , Forand R
Published: May 2006

Extracts of bovine hypothalamus were found to contain a significant level of mitogenic activity when tested in a Swiss 3T3 cell [3H]dThd incorporation assay and in a human umbilical vein endothelial cell growth assay. The mitogenic activity responsible for 3T3 cell activity was purified and characterized as a fibroblast growth factor (FGF)-like mitogen. Neither the biologically active FGF-like mitogen purified from the hypothalamus extracts nor FGF purified from bovine pituitary glands was mitogenic when added to human endothelial cells in vitro, suggesting the presence of more than one mitogen in the hypothalamic extracts. The 3T3 and endothelial cell biological activities of hypothalamic extracts were both found to be inactivated by trypsin, subtilisin, and heat treatment, but were stable to dialysis. The endothelial cell growth factor activity could be efficiently separated from the FGF activity by gel exclusion chromatography. The endothelial cell mitogen possessed a molecular weight of approximately 75,000, whereas that of FGF was approximately 15,000. The endothelial cell growth factor activity was found to be inactivated with reducing agents whereas the 3T3 cell mitogenic activity was stable after incubation with 2-mercaptoethanol. Significant levels of endothelial cell mitogenic activity were also found in extracts of bovine brain and pituitary glands.
Inhibition of polyisoprenoid and glycoprotein biosynthesis causes abnormal embryonic development.
Authors: Carson D D , Lennarz W J
Published: May 2006

Compactin, a potent inhibitor of polyisoprenoid biosynthesis, induces abnormal gastrulation during sea urchin development at concentrations that have no effect on earlier embryonic development or on macromolecular synthesis. Three lines of evidence suggest that the developmental lesion caused by compactin results from inhibition of dolichol biosynthesis and a concomitant inhibition in the biosynthesis of the oligosaccharide chains of N-linked glycoproteins. (i) Embryos cultured in the presence of compactin gastrulate normally when supplemented with dolichol alone, whereas supplementation with cholesterol or coenzyme Q or both does not prevent the compactin-induced developmental lesion. (ii) Exogenously supplemented [3H]dolichol is incorporated into a compound with the chromatographic properties of oligosaccharide-pyrophosphoryldolichol. (iii) Embryos cultured in the presence of compactin exhibit a decreased capacity to synthesize mannose-labeled glycolipids and N-linked glycoproteins. This decrease in synthesis is abolished if the embryos are cultured in the presence of dolichol along with compactin.
Early termination of heterogeneous nuclear RNA transcripts in mammalian cells: accentuation by 5,6-dichloro 1-beta-D-ribofuranosylbenzimidazole.
Authors: Tamm I , Kikuchi T
Published: May 2006

Labeling of RNA in isolated HeLa cell nuclei in vitro reveals an abundance of short RNA chains made by RNA polymerase II. These short chains were initiated prior to isolation of the nuclei. The short abundant chains are increased in amount in nuclei isolated from cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Kinetic evidence indicates that the bulk of the putative heterogeneous nuclear RNA (hnRNA) precursor molecules that are terminated early in vivo are terminated approximately 100-300 nucleotides from sites of initiation. DRB increases the frequency of early termination, but there is a fraction of hnRNA precursor molecules whose elongation is not affected by DRB. Heparin is useful in studies of hnRNA transcription in isolated nuclei because it enhances chain elongation.
Apolipoprotein is responsible for neutralization of xenotropic type C virus by mouse serum
Authors: Kane John P. , Hardman David A. , Dimpfl Joan C. , Levy Jay A.
Published: May 2006

We have shown that the circulating lipoproteins of the mouse contain a potent inhibitor of infectivity of the xenotropic type C virus. This virus neutralization does not involve immunoglobulins or complement. After fractionation of the lipoproteins on the basis of particle size, flotation properties, and electrostatic charge, virus neutralizing activity is found primarily in the triglyceride-rich lipoproteins (predominantly the chylomicrons) and in the HDL
2
subfraction of the high density lipoproteins. In fasted animals, activity resides chiefly in the high density lipoproteins. Neutralization titers increase strikingly during alimentary lipemia in both the lipoproteins of the ρ < 1.006 g/cm
3
fraction and the high density lipoproteins. Increased activity persists in the high density lipoproteins after the lipemia recedes. Virus neutralizing activity is completely eliminated in all fractions by antiserum against high density lipoproteins and, in the triglyceride-rich fractions, by antiserum to murine apolipoprotein B. Complete removal of lipids markedly reduces the neutralizing activity of both classes of lipoproteins. Apolipoproteins delipidated with tetramethylurea retain some activity, which is enhanced by binding to a phospholipid-stabilized triglyceride emulsion and which is abolished by proteolytic digestion. We have demonstrated
in vitro
transfer of activity between high density and very low density lipoproteins of the mouse. These data indicate that xenotropic virus neutralization by normal mouse serum depends upon a protein that transfers among lipoprotein particles in a fashion analogous to the C apolipoproteins of other mammalian species.
Chronic treatment with lithium or desipramine alters discharge frequency and norepinephrine responsiveness of cerebellar Purkinje cells.
Authors: Siggins G R , Schultz J E
Published: May 2006

Cerebellar Purkinje cells were studied by electrophysiological techniques in rats treated chronically with either desipramine (DMI) or lithium chloride given intragastrically. A striking decrement occurred in discharge frequencies of simple spikes and climbing fiber bursts in both groups of animals, similar to the depression produced by iontophoresis of these agents. Chronic treatment with DMI markedly decreased responsiveness to iontophoretically applied norepinephrine (NE), whereas long-term LiCl therapy slightly enhanced response to NE; responses to gamma-aminobutyric acid were unchanged by these treatments. The inhibitory responses to locus ceruleus stimulation were unaffected by chronic LiCl treatment. The effects of these chronic treatments on responsiveness to NE are opposite to the effects these same drugs produce when administered by acute iontophoresis to single cells: DMI then potentiates and LiCl antagonizes noradrenergic responses. These results provide electrophysiological evidence for reciprocal adaptive changes in NE sensitivity, supporting results of biochemical studies.
Binding of
cis
- and
trans
-dichlorodiammineplatinum(II) to the nucleosome core
Authors: Lippard Stephen J. , Hoeschele James D.
Published: May 2006

The binding of the antitumor drug
cis
-dichlorodiammineplatinum(II) and its inactive
trans
isomer with the nucleosome core particle has been investigated. Kinetic studies show that platinum binding increases with incubation time, from a few bound platinum atoms per nucleosome core in the first 0.5 hr to 40-50 after 40 hr. There is no crosslinking or dissociation of nucleosome cores upon platinum binding, as revealed by sedimentation velocity measurements. Electrophoresis through 0.1% sodium dodecyl sulfate/18% polyacrylamide gels after platinum binding reveals striking differences in the DNA and protein band patterns for the two isomers.
cis
-Dichlorodiammineplatinum(II) binds first to the DNA, retarding and spreading its migration in the gel. A comparison study with the 146-base-pair nucleosome core DNA showed the binding to be little affected by the presence of the histone octamer. The
trans
complex, on the other hand, produces DNA—histone and histone—histone crosslinks that only appear for the
cis
isomer after long incubation times. The protein—protein crosslinks were reversed by soaking the gel in cyanide solution to form [Pt(CN)
4
]
-2
. Subsequent two-dimensional gel electrophoresis revealed that
trans
-dichlorodiammineplatinum(II) forms specific crosslinks between histone protein pairs H3 and H2a and H2b and H4 in the nucleosome core. The occurrence of DNA—protein crosslinks was demonstrated by treating the platinum/nucleosome core reaction mixtures with a protease or with nucleases prior to electrophoresis and observing changes in the gel patterns. Platinum was located in the gels through autoradiography using
195m
Pt-labeled complexes. This work clearly demonstrates the greater propensity of
trans
-dichlorodiammineplatinum(II) to form histone—histone and histone—DNA crosslinks compared with the antitumor active
cis
isomer, which binds first to the DNA and only forms crosslinks to the histones when the nucleosome core is heavily loaded with platinum.
Calcineurin: a calcium- and calmodulin-binding protein of the nervous system.
Authors: Klee C B , Crouch T H , Krinks M H
Published: May 2006

The inhibitory protein that binds calmodulin and thus prevents activation of several Ca2+-dependent enzymes by calmodulin is shown to also bind four Ca2+ per mol of protein with high affinity (Kd less than or equal to 10(-6) M). On the basis of its Ca2+- binding properties and its localization to nervous tissue, the inhibitory protein is now called "calcineurin." Calcineurin is composed of two subunits: calcineurin A (61,000 Mr) which interacts with calmodulin in a Ca2+-dependent fashion, and calcineurin B (15,000 Mr) which binds Ca2+. The interaction of calcineurin A with calcineurin B is independent of Ca2+ or Mg2+. The dual interaction of calcineurin A with two different Ca2+-binding components and the high affinity of calcineurin for Ca2+ suggest a possible role for calcineurin in the regulation of free Ca2+ concentrations in the nervous system. Calcineurin may thereby modulate the release and action of neurotransmitters.
A general ellipsoid cannot always serve as a model for the rotational diffusion properties of arbitrarily shaped rigid molecules
Authors: Wegener William A. , Koester Vaughn J. , Dowben Robert M.
Published: May 2006

An ellipsoidally shaped body, or more commonly, an ellipsoid of revolution, is generally assumed to serve as a convenient model for evaluating the rotational diffusion properties of macromolecules. If Perrin's equations for the rotational diffusion coefficients of general ellipsoids can be shown to generate all possible rotational diffusion coefficients, then there would exist at least one equivalent ellipsoidal shape for every arbitrarily shaped rigid body. We investigated the problem by first generating a space,
r
-space, representing all possible ellipsoidal shapes. We then generated another space,
D
-space, representing all possible combinations of rotational diffusion coefficients. We then mapped
r
-space into
D
-space by using Perrin's equations. Ellipsoidal shapes map into diffusion space in a well-defined manner. The mapping is either 1:1, 2:1, or 3:1; several distinctly different regions of
r
-space map onto the same regions of
D
-space. Thus, for some combinations of rotational diffusion coefficients, more than one ellipsoid can be used as a model. Not all of
D
-space is covered by the mapping of
r
-space. Therefore, there are combinations of rotational diffusion coefficients that cannot be generated from ellipsoidally shaped bodies. Several examples of rigid body shapes with nonellipsoidal diffusion properties are described.
Tetrodotoxin-sensitive sodium channels in normal human fibroblasts and normal human glia-like cells.
Authors: Munson R , Westermark B , Glaser L
Published: May 2006

Tetrodotoxin-sensitive sodium channels are detectable in normal human fibroblasts and in "glia-like" cells at appreciable levels when compared to what is observed in established neuronal cell lines in culture. Two- to 3-fold stimulations of sodium influx are observed in the presence of 0.2 mM veratridine and scorpion venom at 0.1 mg/ml. Tetrodotoxin (2 microM) inhibits the observed stimulation of sodium influx. Previous work has indicated that these neurotoxins act on the voltage-sensitive sodium ionophore of excitable cells, and the presence of such channels in cells generally considered nonexcitable raises questions regarding both the uniqueness of this ionophore as a property of excitable cells and the origin of the cells generally described as fibroblasts.
Presence of calmodulin in Tetrahymena.
Authors: Jamieson G A , Vanaman T C , Blum J J
Published: May 2006

Ca-dependent affinity chromatography on phenothiazine-Sepharose 4B has been used to isolate a pure protein from the ciliate Tetrahymena pyriformis. This protein has been identified as calmodulin by demonstrating three of the Ca-dependent activities attributed to calmodulins. Tetrahymena calmodulin also has physicochemical properties similar to those of the previously characterized mammalian, coelenterate, and plant proteins, except for a lower molecular weight (15,000) and slightly different CNBr fragments compared to bovine brain calmodulin. Calmodulin is a constituent of demembranated Tetrahymena cilia from which it can be extracted with the crude dynein fraction. Sucrose density gradient fractionation indicated its presence in fractions containing the 14S dynein ATPase. It is concluded that the essential properties of calmodulin have been highly conserved during much of eukaryotic evolution, and it is suggested that calmodulin plays a role in the control of ciliary motility in Tetrahymena.
Characterization of a mutator gene in Chinese hamster ovary cells.
Authors: Meuth M , L'Heureux-Huard N , Trudel M
Published: May 2006

We have recently reported the isolation of a class of mutants (called thy-) that is both resistant to arabinosyl cytosine and auxotrophic for thymidine. thy- mutants have a 5- to 10-fold elevated pool of dCTP and are deficient in the synthesis of dTTP as an apparent consequence of a single mutation in the gene for ribonucleoside-diphosphate reductase (2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1). Here we show that three independent thy- lines have a 5- to 50-fold higher frequency and rate of spontaneous mutation for two genetic markers, 6-thioguanine resistance and ouabain resistance. The higher rate of mutation is site specific because two other genetic markers, reversion of proline auxotrophy to proline prototrophy and emetine resistance, are unaffected. Ouabain- and 6-thioguanine-resistant mutations occur at a much lower rate in revertants of thy- to the wild-type state, so the increased rate of mutation is the consequence of the thy- mutation. Both the increased mutational rate and the increased intracellular pools of dCTP are dominant or codominant in hybrid cells, and alterations of the ratio of the pools of dCTP to dTTP in thy- 49 produce corresponding changes in the rate of mutation. Thus, thy- is a mutator gene in Chinese hamster ovary cells, apparently as a consequence of the imbalance of deoxynucleoside triphosphate pools created by the expanded pool of dCTP.
Tumorigenicity of embryonal carcinoma as an assay to study control of malignancy by the murine blastocyst.
Authors: Pierce G B , Lewis S H , Miller G J , Moritz E , Miller P
Published: May 2006

A bioassay, based on tumorigenicity, has been developed to determine the mechanism whereby the blastocyst of the mouse controls malignant expression of embryonal carcinoma. The assay is based upon the incidence of tumors obtained when known numbers of cells of the 402AX strain of embryonal carcinoma are injected into strain 129 mice, compared to the incidence obtained when the same number of embryonal carcinoma cells are incorporated into Swiss-Webster blastocysts that are then injected in strain 129 animals. The results indicate that the blastocyst can regulate one embryonal carcinoma cell consistently; it may have a slight effect on three, but it cannot regulate four or five of them. The position of the embryonal carcinoma cell in the blastocyst is important. Regulation occurs if the embryonal carcinoma cell is placed in the blastocoele cavity, but enhancement of tumorigenicity is obtained if it is placed between the zona pellucida and the trophectoderm. By contrast, the blastocyst is unable to regulate a single B-16 melanoma cell placed in the blastocoele cavity, indicating a degree of specificity for the regulatory process.
Use of a lipophilic cation for determination of membrane potential in neuroblastoma-glioma hybrid cell suspensions
Authors: Lichtshtein David , Kaback H. Ronald , Blume Arthur Joel
Published: May 2006

Neuroblastoma-glioma hybrid cells (NG108-15) in suspension accumulate the permeant lipophilic cation [
3
H]tetraphenylphosphonium (TPP
+
) against a concentration gradient. The steady-state level of TPP
+
accumulation is about twice as great in physiological media of low K
+
concentration (i.e., 5 mM K
+
/135 mM Na
+
) than in a medium of high K
+
concentration (i.e., 121 mM K
+
/13.5 mM Na
+
). The latter manipulation depolarizes the NG108-15 plasma membrane and indicates that the resting membrane potential (ΔΨ) is due primarily to a K
+
diffusion gradient (K
in
+
→ K
out
+
). TPP
+
accumulation is time and temperature dependent, achieving a steady state in 15-20 min at 37°C, and is a linear function of cell number and TPP
+
concentration (i.e., the concentration gradient is constant). The difference in TPP
+
accumulation in low and high K
+
media under various conditions has been used to calculate mean (±SD) ΔΨ values of -56 ± 3, -63 ± 4, and -66 ± 5 mV at 26, 33, and 37°C, respectively. Importantly, these values are virtually identical to those obtained by direct electrophysiological measurements made under the same conditions. TPP
+
accumulation is abolished by the protonophore carbonylcyanide-
m
-chlorophenylhydrazone, whereas the neurotoxic alkaloid veratridine diminishes uptake to the same level as that observed in high K
+
media. In addition, the effect of veratridine is dependent upon the presence of external Na
+
and is blocked by tetrodotoxin. The steady-state level of TPP
+
accumulation is enhanced by monensin, indicating that this ionophore induces hyperpolarization under appropriate conditions. Finally, ouabain has essentially no effect on the steady-state level of TPP
+
accumulation in short-term experiments, suggesting that Na
+
,K
+
-ATPase activity makes little contribution to the resting potential in these cells. Because many of these observations are corroborated by intracellular recording techniques, it is concluded that TPP
+
distribution measurements can provide a biochemical method for determining membrane potentials in populations of cultured neuronal cells.