bluuebunny/crossref_metadata_2025 huggingface dataset:
Semisynthetic horse heart [65-homoserine]cytochrome
c
from three fragments
Authors: Boon Peter J. , Tesser Godefridus I. , Nivard Rutger J. F.
Published: May 2006

Horse heart cytochrome
c
was treated with methylsulfonylethyloxycarbonyl succinimide (Msc-ONSu) to give fully

N
ε

-protected cytochrome
c
. Treatment of this derivative with a hard base for 15 sec regenerated the native tetrahectapeptide chain. CNBr degradation of the protected compound produced three fragments bearing only protective Msc functions on ε-amino groups. The fragment comprising the sequence 81-104 was isolated from the mixture and acylated with
N
-hydroxysuccinimidyl-
t
-butyloxycarbonyl-L-methioninate. The resulting pentacosapeptide derivative was partially deprotected by treatment with acid and condensed in good yield (65%) with fully synthetic
N

α
66

,
N

ε
72,73,79

- tetra-Msc-cytochrome-
c
-(66-79)-tetradecapeptide azide. This pathway is preferred because the
penta
decapeptide azide derivative 66-80 acylated the

N
ε

-protected tetracosapeptide sequence 81-104 in an unpredictable manner. Subsequent treatment of the product with a base produced unprotected semisynthetic cytochrome-
c
-(66-104)-nonatriacontapeptide, which is known to undergo acylation by unprotected [Hse
65
]cytochrome-
c
-(1-65)-pentahexacontapeptide lactone. The high specificity of this condensation is ascribed to “conformation direction.” Semisynthetic [Hse
65
]cytochrome
c
thus prepared reacts like native cytochrome
c
with a succinate cytochrome
c
reductase preparation and with cytochrome
c
oxidase (ferrocytochrome
c
:oxygen oxidoreductase, EC 1.9.3.1). This semisynthetic strategy may provide a rapid route for the production of cytochrome
c
analogs modified in the highly conservative sequence 66-80.
Thermosensory transduction in Escherichia coli: inhibition of the thermoresponse by L-serine.
Authors: Maeda K , Imae Y
Published: May 2006

Information processing of the thermoresponse in Escherichia coli was compared with that of the chemoresponse. Competition experiments between various chemical stimuli and the thermal stimulus showed that only L-serine was a potent inhibitor of the thermosensory transduction. The concentration of L-serine necessary for complete inhibition of the thermoresponse was about 0.1 mM. L-Serine at this concentration did not inhibit chemoresponses to many amino acids. Pleiotropic aspartate-taxis mutants (tar) showed normal thermoresponse but pleiotropic serine-taxis mutants (tsr) showed decreased or almost no thermoresponse. These results suggest that the thermosensory transducing system in E. coli has an intimate interaction with the chemosensory transducing pathway specific for L-serine. A simple model for the thermosensory transduction is discussed.
Quaternary structure of the ribosomal 30S subunit: model and its experimental testing.
Authors: Spirin A S , Serdyuk I N , Shpungin J L , Vasiliev V D
Published: May 2006

In considering the structure of the 30S subunit of the Escherichia coli ribosome, we have assumed that: (i) all or almost all the proteins within the 30S particle are compact and globular, as recently shown for the isolated proteins S4, S7, S8, S15, and S16 in solution [Serdyuk, I.N., Zaccai, G. & Spirin, A.S. (1978) FEBS Lett. 94, 349-352]; (ii) the RNA within the 30S particle has approximately the same specific V-like or Y-like shape that was demonstrated for the isolated 16S RNA in a compact conformation [Vasiliev, V.D., Selivanova, O.M. & Koteliansky, V.E. (1978) FEBS Lett. 95, 273-276]. From these assumptions and using the numerous data reported on neighboring ribosomal proteins, we have constructed a model of the quaternary structure of the ribosomal 30S subunit. The model has been tested by calculation of the theoretical curves of neutron scattering at different contrasts, as well as those of x-ray scattering, and their comparison with the experimental scattering curves for E. coli 30S particles. It has been found that the calculated scattering curves for the model practically coincide with the experimental scattering curves for the 30S particles in the range of Bragg distances down to 40-55 A. The scattering curves calculated for several three-dimensional patterns of arrangement of the 30S subunit proteins proposed earlier have been shown to be inconsistent with the experiments.
Cloning and nucleotide sequence of DNA coding for bovine preproparathyroid hormone.
Authors: Kronenberg H M , McDevitt B E , Majzoub J A , Nathans J , Sharp P A , Potts J T , Rich A
Published: May 2006

We have cloned in Escherichia coli a DNA copy of mRNA coding for bovine preproparathyroid hormone. Double-stranded DNA was inserted into the Pst I site in plasmid pBR322 by using the poly(dG)-poly(dC) homopolymer extension technique to join the DNA molecules. Recombinant plasmids coding for preproparathyroid hormone were identified by the plasmid's ability to arrest specifically the translation of preproparathyroid hormone mRNA. The nucleotide sequence of the largest recombinant was determined by using both chemical and enzymatic techniques. The parathyroid insert contains 470 nucleotides--102 nucleotides from the 5' noncoding region of the mRNA, 345 nucleotides representing the entire coding region, and 23 nucleotides from the 3' noncoding region. The coding sequence clarifies the hormone's amino acid sequence, which has been disputed. Codon usage is discussed.
Lateral diffusion of surface immunoglobulin, Thy-1 antigen, and a lipid probe in lymphocyte plasma membranes
Authors: Dragsten Paul , Henkart Pierre , Blumenthal Robert , Weinstein John , Schlessinger Joseph
Published: May 2006

Fluorescence photobleaching recovery was used to measure the lateral diffusion coefficient and mobile fraction of surface immunoglobulin (sIg), Thy-1 antigen, and a lipid probe in the plasma membrane of mouse lymphocytes. The lipid probe (3,3′-dioctadecylindocarbocyanine) had a mean (±SD) diffusion coefficient of (1.7 ± 0.3) × 10
-8
cm
2
/sec, with essentially all of the probe mobile in the membrane. We detected little or no effect on the diffusion of this probe due to the presence of microvilli. Its diffusion was slightly restricted in capped regions. No differences in lipid probe mobility were detected between T and B cells. Fifty to 90% of the detectable sIg and Thy-1 antigen was free to move in the plane of the membrane with diffusion coefficients of ≈3 × 10
-10
cm
2
/sec; the remainder was immobile. Crosslinking of sIg with anti-Ig antibodies (in the presence of azide to inhibit capping) completely immobilized sIg at high concentrations but failed to do so at low concentrations. Thy-1 antigen could not be immobilized with an IgG rabbit anti-mouse brain reagent without an additional layer of crosslinking antibody. In parallel labelings (in the absence of azide), capping of sIg and Thy-1 antigen was observed only under crosslinking conditions sufficient to immobilize the membrane antigen. Sodium azide, colchicine, and cytochalasin B had no measurable effect on lipid probe, sIg, or Thy-1 diffusion.
Synthesis and turnover of globin mRNA in murine erythroleukemia cells induced with hemin
Authors: Lowenhaupt Ky , Lingrel Jerry B.
Published: May 2006

When murine erythroleukemia (MEL) cells are induced with hemin, they carry out several early functions of the erythroid program. However, they do not become committed to terminal differentiation nor do they become benzidine positive. This is in contrast to MEL cells induced with dimethyl sulfoxide (Me
2
SO) which undergo a more complete program of erythroid differentiation. In order to determine the relationship between commitment and various events in the erythroid program, we compared the induction of MEL cells with hemin and with Me
2
SO. The amount of globin mRNA accumulated in inducing MEL cells and the rate of its synthesis and turnover were quantitated. Although MEL cells induced with hemin accumulated significantly less globin mRNA than did cells induced with Me
2
SO, the rate of synthesis of globin mRNA was the same in fully induced cells, irrespective of inducer. Therefore, there is no evidence that induction with hemin produces an early program that is different or altered from that which is part of Me
2
SO induction. MEL cells induced with Me
2
SO specifically destabilize their globin mRNA after 4 days of induction. This raises the question of whether this destabilization of globin mRNA is an independently programmed late event, as suggested by the time of its occurrence, or, alternatively, whether it might be the inevitable consequence of an early event(s). For instance, destabilization might be linked to the synthesis or translation of globin mRNA. Because MEL cells induced with hemin do not destabilize their globin mRNA, we have concluded that this turnover of globin mRNA is a late event, occurring only in a committed cell population.
Nucleosome core histone complex isolated gently and rapidly in 2 M NaCl is octameric.
Authors: Philip M , Jamaluddin M , Sastry R V , Chandra H S
Published: May 2006

A relatively stable specific complex of the chromatin core histones H2A, H2B, H3, and H4 has been obtained in 2 M NaCl/25 mM sodium phosphate buffer, pH 7.0. The histone core complex has an apparent specific volume of 0.73 ml/g. Its sedimentation coefficient was dependent on rotor speed (angular velocity, omega) and attained different stable values at low and high rotor speeds. The drop in sedimentation coefficient occurred sharply between omega 2 values of about 9 x 10(6) and 1.1 x 10(7) (radians/sec)2. The s020,w corresponding to zero angular velocity (1 atmosphere pressure) was 6.6 S +/- (SEM) 0.1 S. At high rotor speeds the value decreased to 3.8 S +/- 0.1 S. The core complex has a diffusion coefficient, D20,w, of 5.4 x 10(-7) cm2/sec and a molecular weight of 108,000 +/- (SD) 2500.
Biogenesis of intestinal plasma membrane: Posttranslational route and cleavage of sucrase—isomaltase
Authors: Hauri Hans-Peter , Quaroni Andrea , Isselbacher Kurt J.
Published: May 2006

The biosynthesis
in vivo
of rat intestinal sucrase—isomaltase [a complex of sucrose α-glucohydrolase, EC 3.2.1.48, and oligo-1,6-glucosidase (dextrin 6-α-D-glucanohydrolase), EC 3.2.1.10] has been studied by following the incorporation of L-[6-
3
H]fucose into the enzyme with time. Immunoprecipitation of sucrase—isomaltase from Triton-X-100-solubilized Golgi or basolateral membranes and subsequent polyacrylamide gel electrophoresis revealed the presence of an immunoreactive glycoprotein with an apparent molecular weight approximately twice that of the separated sucrase—isomaltase subunits, but no active subunits were found in these membranes. This glycoprotein was also found in the microvillus membrane in addition to the subunits of sucrase—isomaltase. Kinetic studies showed a maximal labeling of this glycoprotein in Golgi membranes at 15 min, in basolateral membranes at 30 min, and in microvillus membranes at 45 min and a half-life of less than 30 min in each membrane. However, the radioactivity of the sucrase—isomaltase subunits in the microvillus membrane reached a plateau after 60 min. These data suggest that sucrase—isomaltase is synthesized as a one-chain polypeptide precursor that is split into the subunits after its transfer to the microvillus membrane. Elastase (EC 3.4.21.11), but not trypsin (EC 3.4.21.4) or α-chymotrypsin (EC 3.4.21.1), split the putative precursor into two polypeptides that had electrophoretic behaviors similar to those of the active enzyme subunits. These studies suggest that pancreatic proteases may play an important role in the late posttranslational processing of sucrase—isomaltase
in vivo
.
Immunocytochemical localization of epoxide hydrase in hyperplastic nodules induced in rat liver by 2-acetylaminofluorene.
Authors: Novikoff A B , Novikoff P M , Stockert R J , Becker F F , Yam A , Poruchynsky M S , Levin W , Thomas P E
Published: May 2006

A knowledge of the biological characteristics of carcinogen-induced hyperplastic nodules of rat liver may be important in the understanding of cancer development. Although its biological role remains to be elucidated, the level of microsomal epoxide hydrase (epoxide hydrolase, EC 3.3.2.3) is 5- to 7-fold greater in hyperplastic nodules nodules induced by feeding the hepatocarcinogen 2-acetylaminofluorene than in liver of control rats. After removal of the carcinogen from the diet, the high level of the enzyme is maintained in those nodules that persist and in the hepatocellular carcinomas that subsequently develop. The availability of antibody to the epoxide hydrase made it possible to use electron microscopic immunocytochemistry to localize this enzyme in the cells of hyperplastic nodules. The immunocytochemical procedure provides direct visual evidence for the presence of this enzyme in smooth endoplasmic reticulum and also in rough endoplasmic reticulum (including the nuclear envelope) of the nodule's parenchymal cells.
Efficient transformation of
Neurospora crassa
by utilizing hybrid plasmid DNA
Authors: Case Mary E. , Schweizer Michael , Kushner Sidney R. , Giles Norman H.
Published: May 2006

An efficient transformation system has been developed for
Neurospora crassa
that uses spheroplasts and pVK88 plasmid DNA. pVK88 is a recombinant
Escherichia coli
plasmid carrying the
N. crassa qa-2
+
gene which encodes catabolic dehydroquinase (3-dehydroquinate hydro-lyase, EC 4.2.1.10) and is part of the
qa
gene cluster. The recipient strain carries a stable
qa-2
-
mutation and an
arom-9
-
mutation, thus lacking both catabolic and biosynthetic dehydroquinase activities. Transformants were selected as colonies able to grow in the absence of an aromatic amino acid supplement. These colonies were
qa-2
+
and had normal levels of catabolic dehydroquinase. DNA·DNA hybridization evidence with appropriate labeled probes indicates clearly that in some instances transformation involves the integration of bacterial plasmid sequences together with the
qa-2
+
gene into the
N. crassa
genome. On the basis of genetic, enzyme assay, and DNA hybridization data, at least three types of transformation events can be distinguished: (
i
) replacement of the
qa-2
-
gene by the
qa-2
+
gene without any effect on the expression of the other genes in the
qa
cluster, (
ii
) linked insertion of a normal
qa-2
+
gene accompanied by inactivation of the adjacent
qa-4
+
gene, and (
iii
) insertion of a normal
qa-2
+
gene at an unlinked site in the
N. crassa
genome. This newly integrated
qa-2
+
genetic material is inherited in a typical Mendelian fashion. A low level of transformation has also been obtained by using linear total
N. crassa
DNA. Two such
qa-2
+
transformants are unlinked to the
qa-2
-
gene of the recipient.
Involvement of gangliosides in lymphocyte stimulation.
Authors: Spiegel S , Ravid A , Wilchek M
Published: May 2006

N4-Dinitrophenyl-L-2,4-diaminobutyric acid hydrazide was attached covalently to the aldehyde groups produced by periodate oxidate of bovine brain gangliosides and it was incorporated into mature thymocytes. High concentrations of antibodies to dinitrophenyl (DNP) or lower concentrations followed by protein A agglutinated the cells and stimulated DNA synthesis. Incubation of the thymocytes containing DNP-modified ganglioside with rabbit anti-DNP and fluorescein-labeled anti-rabbit IgG resulted in the formation of uniform rings on the cell surface, which subsequently developed into patches and caps. These results indicate that gangliosides may play a primary role in the lymphocyte activation process.
Growth of complex systems can be related to the properties of their underlying determinants
Authors: Savageau Michael A.
Published: May 2006

Growth—increase in size, number, or amount—in many cases appears to follow simple empirical laws. Such laws have been noted in a wide variety of fields for many years. Until now these laws have never been related to the underlying determinants of these systems. By starting with fundamental properties of the component mechanisms in such systems, one can derive a basic growth equation for which the well-known laws of growth are special cases.
Differentiation as a requirement for simian virus 40 gene expression in F-9 embryonal carcinoma cells.
Authors: Segal S , Khoury G
Published: May 2006

Infection of differentiated mouse embryo cells by simian virus 40 (SV40) leads to the production of the early mRNAs and the tumor (T) antigens that they encode. In contrast, undifferentiated F-9 murine teratocarcinoma cells do not support these early stages of the SV40 cycle. This block results from the inability to accumulate stable processed early SV40 mRNAs. It has recently been shown that vitamin A and its derivatives can induce in vitro differentiation of stem cells. Undifferentiated F-9 cells, upon treatment with a low concentration of retinoic acid, exhibited pronounced morphologic changes as well as the appearance of the H-2 surface antigens. After differentiation, the susceptibility of F9 cells to SV40 infection could be demonstrated by the appearance of large T and small T antigens, as shown by immunofluorescence and immunoprecipitation. Furthermore, SI nuclease mapping of early SV40 transcripts confirmed the presence of the two spliced early mRNAs. These results indicate that the undifferentiated F-9 stem cells contain the genetic information needed for generating stable processed early SV40 mRNAs but are blocked in the production of functional species.
Epidermal growth factor stimulation of DNA synthesis is potentiated by compounds that inhibit its clustering in coated pits
Authors: Maxfield Frederick R. , Davies Peter J. A. , Klempner Lev , Willingham Mark C. , Pastan Ira
Published: May 2006

We have used inhibitors of receptor-mediated endocytosis to investigate the mechanism and function of epidermal growth factor uptake by cultured cells. When rhodamine-labeled epidermal growth factor is bound to cell surface receptors on confluent monolayers of BALB/c 3T3 cells, it rapidly collects in cell surface clusters and is internalized. The clustering of occupied receptors requires Ca
2+
and is inhibited by primary alkylamines; both of these properties are shared by the enzyme transglutaminase (R-glutaminyl-peptide:amine γ-glutamyl-yltransferase, EC 2.3.2.13). In Chinese hamster ovary cell extracts, methylamine inhibits 25-50% of the transglutaminase activity with a
K
i
of 0.2 mM, and it inhibits the remaining transglutaminase activity with a
K
i
of 20 mM. Clustering is almost completely inhibited by 10 mM methylamine. The polypeptide antibiotic bacitracin inhibits clustering of rhodamine-labeled epidermal growth factor or α
2
-macroglobulin at 0.7 mM, and it inhibits approximately 40% of the transglutaminase activity in Chinese hamster ovary cells with a
K
i
of 0.03 mM. Fluorescent ligands bound to cell surface receptors in the presence of bacitracin form clusters within 30 min after bacitracin is removed from the culture medium. These results indicate that a transglutaminase-like enzyme may be required for the clustering and subsequent internalization of occupied receptors. The effects of 10 mM methylamine and 0.7 mM bacitracin on epidermal growth factor stimulation of DNA synthesis were examined. The stimulation of DNA synthesis by epidermal growth factor was increased 2- to 7-fold in the presence of methylamine or bacitracin. Alone, methylamine or bacitracin increased DNA synthesis 1.1- to 3-fold. The stimulation of DNA synthesis resulting from the simultaneous presence of the hormone and the clustering inhibitor was always greater than the sum of the stimulations produced by the hormone and the clustering inhibitors alone. The potentiation of epidermal growth factor activity by clustering inhibitors suggests that the hormone acts at the cell surface. We propose that rapid internalization of occupied receptors via coated pits may be a mechanism to limit the response to hormones.
Prevention of genetic anemias in mice by microinjection of normal hematopoietic stem cells into the fetal placenta
Authors: Fleischman Roger A. , Mintz Beatrice
Published: May 2006

Mice homozygous for mutant genes at the
W
locus have a marked macrocytic anemia that is fatal in some genotypes. The defect is believed to originate in the developmentally pluripotent hematopoietic stem cell population. Anemia is first grossly manifest on day 13 of gestation, when the liver is the chief hematopoietic organ. The known paucity of blood-forming foci in livers of homozygotes and the limited formation of their erythrocytes suggested that such fetuses—unlike normal ones—might have conditions favorable for
in utero
seeding of genetically normal hematopoietic tissue. If this were accomplished before day 13, the anemia might essentially be prevented, or at least substantially mitigated, and normalcy soon achieved by cell selection. This proved to be the case. Allogeneic normal fetal liver cells were microinjected into the blood vessels of the fetal placenta on day 11 of gestation. Of eight mutant homozygotes born from segregating matings, six (four
W/W
, two

W
v
/W
v

) were successfully populated with donor cells. Strain-specific hemoglobin markers demonstrated replacement of the erythroid lineage with the normal type, the rate of substitution being more rapid in the
W/W
(ordinarily more anemic) recipients. Strain-specific isozyme differences revealed that white blood cells were also replaced. Thus, the initial selective pressure, hence the
W
-mutant phenotypic lesion, must have occurred at the pluripotent stem cell stage. The animals remained immunologically tolerant of the donor cells and no graft-versus-host reaction occurred. The early introduction of hematopoietic cells differing genetically from all the other tissues of the animal provides possibilities for tracing normal hematopoietic lineages
in vivo
, for analyzing cell and tissue interactions, such as those between lymphocytes and thymus, and for clarifying the etiology of other blood or immune insufficiencies or malignancies.
Regional assignment of the steroid sulfatase—X-linked ichthyosis locus: Implications for a noninactivated region on the short arm of human X chromosome
Authors: Mohandas T. , Shapiro L. J. , Sparkes R. S. , Sparkes M. C.
Published: May 2006

The expression of steroid sulfatase (SS; sterol-sulfatase; sterol-sulfate sulfohydrolase, EC 3.1.6.2), a microsomal enzyme that catalyzes the hydrolysis of a variety of 3β-hydroxysteroid sulfates, was evaluated in mouse—human hybrid clones. The mouse parental line, A9, was found to have little SS as determined by activity measurements. Human SS can be separated from mouse SS by electrophoresis. Two human fibroblast lines, one carrying an X/13 translocation [46,X,t(X;13)(p22;q12)] and the other carrying an X/20 translocation [46,X,t(X;20)(Xp20q;Xq20p)] were used as the human parental lines. Several independently derived hybrid clones from each of the two fusion experiments were analyzed for the expression of human SS by activity measurements and electrophoresis. Cytogenetic analyses were done on these hybrid clones at the same passage level. The results showed that the expression of human SS in these cell hybrids was concordant only with the presence of the distal half (p22→pter) of the short arm of the human X chromosome, thus assigning the locus for SS to Xp22→Xpter. Earlier studies have shown that the deficiency of SS is the basis for the dermatologic condition X-linked ichthyosis, the gene for which is known to be located approximately 10 centimorgans from the Xg blood group locus. The localization of
SS
on the X chromosome indicates that
Xg
locus may be on the short arm of X and possibly on its distal half. The
Xg
locus is thought to escape X-inactivation in man, and recent investigations suggest that the
SS
locus also escapes X-inactivation. Our results thus provide evidence for the location of an apparently noninactivated site on the distal half of the short arm of the human X-chromosome that contains the locus for
SS
and possibly the
Xg
locus.
Disassembly of viral membranes by complement independent of channel formation.
Authors: Esser A F , Bartholomew R M , Jensen F C , Müller-Eberhard H J
Published: May 2006

We have compared the effects of the complement membrane attack complex (MAC), nystatin, and melittin on the envelope of murine leukemia viruses to determine if channel formation alone is sufficient to cause membranolysis. Nystatin is a channel former and mellitin is not, although both are hemolytic. Whereas MAC and melittin disintegrated the viral membrane, nystatin had no effect on morphology, integrity, and infectivity of the virus. Incorporation of the antibiotic into the viral membranes was demonstrated by measurements of the characteristic fluorescence of nystatin in membranes and the dose-dependent increase in viral density after uptake of the antibiotic. The density of nystatin was measured to be 1.26-1.27 g/cm3. Proof for the formation of functional nystatin channels was obtained by light scattering measurements. Exposure of untreated virus to hypotonic conditions increased viral light scattering because of osmotic swelling but otherwise had no effect on the integrity of the virus. Nystatin channel formation abolished the light scattering change, showing that the antibiotic had impaired the viral permeability barrier. We interpret these results to indicate that virolysis by MAC is not caused by channel formation and, conversely, in the absence of colloid-osmotic effects, channel formation by itself is not sufficient to disassemble a viral membrane.
Allometric morphogenesis of complex systems: Derivation of the basic equations from first principles
Authors: Savageau Michael A.
Published: May 2006

Allometric morphogenesis is the generation of form or pattern as a result of differential growth according to power-law relationships among the elements of a complex system. This phenomenon has been noted in a variety of fields for many years, in some cases for centuries, and yet it has never been related to the underlying determinants of the systems. By starting with fundamental properties of the component mechanisms in such systems, one can derive a basic growth equation from which the well-known law of allometric morphogenesis follows naturally.
Phosphorylation of specific, distinct proteins in synaptosomes and axons from squid nervous system
Authors: Pant Harish C. , Pollard Harvey B. , Pappas George D. , Gainer Harold
Published: May 2006

Synaptosomes and axons from squid were incubated with [γ-
32
P]ATP or [
32
P]orthophosphate and specific, distinct proteins were found to be labeled in each preparation. In axoplasm, only the major 200,000
M
r
neurofilament protein and a specific protein of ≈400,000
M
r
were labeled, as reported previously [Pant, H. C., Shecket, G., Gainer, H. & Lasek, R. J. (1978)
J. Cell Biol.
78, R23-R27]. These results were independent of whether the cosubstrates were
32
PO
4
2-
or [γ-
32
P]ATP. However, synaptosomes lacked the 200,000
M
r
neurofilament protein and several lower molecular weight proteins were labeled instead, the most prominent being a 47,000
M
r
species. [γ-
32
P]ATP was much more effective in labeling the 47,000
M
r
species than
32
PO
4
2-
. Synaptosomes also contained a distinct 250,000
M
r
protein species which, however, was not labeled.

The protein kinase activity in synaptosomes was sensitive to various pharmacological agents, depending on whether the labeled phosphate came directly from ATP or orthophosphate. Carbonyl cyanide
p
-trifluoromethoxyphenyl hydrazone, a mitochondrial H
+
uncoupler, almost completely inhibited incorporation of
32
P into protein with
32
PO
4
2-
as cosubstrate, as expected, but produced only 32% inhibition with [γ-
32
P]ATP as cosubstrate. The activity could be augmented by incubating synaptosomes in a calcium-free medium and could be suppressed by increasing intrasynaptosomal Ca
2+
with A23187, a Ca
2+
ionophore. The latter effect was more prominent with
32
PO
4
2-
than with [γ-
32
P]ATP as cosubstrate. Depolarizing agents such as veratridine and high K
+
also suppressed activity, and the veratridine effect was completely reversed by tetrodotoxin or by omission of Ca
2+
when [γ-
32
P]ATP was used, and partially reversed when
32
PO
4
2-
was used. We conclude that the morphological transformation of an axon into a terminal is accompanied by significant changes in protein and phospho-protein composition that may be related to synaptic transmission.
13C nuclear magnetic resonance studies of anaerobic glycolysis in suspensions of yeast cells.
Authors: den Hollander J A , Brown T R , Ugurbil K , Shulman R G
Published: May 2006

Anaerobic glycolysis in Saccharomyces cerevisiae has been studied by 13C NMR at 90.5 MHz. [1-13c]Glucose and [6-13C]glucose were fed to suspensions of yeast cells. Time courses for concentration changes of the starting material, of courses for concentration changes of the starting material, of the intermediate fructose 1,6-bisphosphate (Fru-P2), and of the end products, ethanol and glycerol, have been followed with 1-min time resolution. The glucose uptake was well fitted by a Michaelis-Menten model, assuming competition of alpha- and beta-glucose for the same site. The Km for the uptake was found to be 10 mM for beta-glucose and 5 mM for alpha-glucose. The concentration of Fru-P2 showed an initial oscillation before it reached a co,stant level. The 13C label, introduced only as [-13C]- or [6-13C]glucose, was observed in Fru-P2 in both the C1 and C6 positions, simultaneously. From the relative intensities of the C1 Fru-P2 and C6 Fru-P2 peaks in the presence of [1-13C]- and [6-13C]glucose, in vivo kinetic information was obtained about the aldolase-triosephosphate isomerase triangle. We found that under the conditions of these experiments the ratios of backward to forward velocities through aldolase and triosephosphate isomerase were 0.9 +/- 0.1 and 0.8 +/- 1, respectively, indicating they were close to equilibrium.
DNA-directed in vitro synthesis of proteins involved in bacterial transcription and translation.
Authors: Zarucki-Schulz T , Jerez C , Goldberg G , Kung H F , Huang K H , Brot N , Weissbach H
Published: May 2006

The in vitro synthesis of elongation factor (EF)-Tu (tufB), the beta beta' subunits of RNA polymerase, ribosomal proteins L10 and L12 directed by DNA from the transducing phage lambda rifd 18, EF-Tu (tufA), EF-G, and the alpha subunit of RNA polymerase directed by DNA from the transducing phage lambda fus3 has been investigated in a crude and a partially defined protein-synthesizing system. Proteins L10 and L12 are synthesized in the partially defined system almost as well as in the crude system. However, the synthesis of EF-Tu, EF-G, and the alpha and beta beta' subunits of RNA polymerase is far less efficient in the partially defined system. An active fraction that stimulates the synthesis of these latter proteins has been obtained by fractionation of a high-speed supernatant on DEAE-cellulose. Because previous studies showed that this fraction (1 M DEAE salt eluate) contains a protein, called L factor, that stimulates beta-galactosidase synthesis in vitro, L factor was tested for activity. Although L factor stimulates the synthesis of the beta beta' subunits, it has little or no effect on the in vitro synthesis of the other products studied. In the present experiments, the ratio of L12/L10 and of EF-Tu (tufA)/EF-G formed is 4-6. These values are consistent with in vivo results.
Mutational analysis of the simian virus 40 replicon: pseudorevertants of mutants with a defective replication origin.
Authors: Shortle D R , Margolskee R F , Nathans D
Published: May 2006

The circular genome of simian virus 40 is a model mammalian replicon, containing a unique origin of replication (ori) and coding for a protein (SV40 T antigen) known to be involved in initiation of viral DNA replication and to bind in vitro to the origin region. Mutations within the ori sequence lead to defective viral DNA replication and the formation of small viral plaques after infection of a cell monolayer. Second-site revertants (pseudorevertants) of ori mutants were isolated by random local mutagenesis of mutant DNA followed by transfection of cultured cells and the selection of large plaques. In each case, reversion of the plaque phenotype was associated with an increased rate of viral DNA replication. The second-site mutations that suppressed the replication defects were localized by in vitro recombination or marker rescue experiments to the gene for T antigen. Their map positions differ from those of previously described T antigen mutants, possibly reflecting a specific ori-binding domain of T antigen. From these results we infer that T antigen interacts with the ori signal during virus development as it does in vitro and that this interaction regulates the rate of viral DNA replication.
Molecular cloning of Moloney murine sarcoma virus: arrangement of virus-related sequences within the normal mouse genome.
Authors: Tronick S R , Robbins K C , Canaani E , Devare S G , Andersen P R , Aaronson S A
Published: May 2006

The unintegrated circular DNA form of Moloney murine sarcoma virus (MSV) has been cloned in bacteriophage lambda. Discrete deletions in the viral genome were shown to occur during propagation of recombinant phage in Escherichia coli. Heteroduplex and restriction enzyme analyses indicated the deletion of tandemly repeated sequences within certain of the cloned MSV DNA inserts. Cloned MSV DNA was used to prepare a probe composed of its acquired cellular (src) sequences, shown previously to be necessary for MSV transformation. Analysis of EcoRI digests of normal mouse cellular DNA revealed the presence of a single 14-kilobase-pair fragment containing these sequences which lacked contiguity with endogenous type C helper viral information of the same cells. Thus, the sarcoma virus-specific sequences of MSV are represented within the normal mouse genome in a manner analogous to that of a cellular gene.
Transport of gamma-glutamyl amino acids: role of glutathione and gamma-glutamyl transpeptidase.
Authors: Griffith O W , Bridges R J , Meister A
Published: May 2006

This work relates to the hypothesis that one of the mechanisms that mediates amino acid translocation across cell membranes involves the action of membrane-bound gamma-glutamyl transpeptidase on intracellular glutathione and extracellular amino acids to form gamma-glutamyl amino acids. According to this idea, the latter are translocated into the cell where the gamma-glutamyl moiety is removed to yield free amino acids. Previous studies in this laboratory showed that intracellular glutathione is translocated out of many cells. We have now directly examined the transport of gamma-glutamyl amino acids into tissues in the mouse by use of the model substrate L-gamma-glutamyl-L-[14C]methionine sulfone. Of 11 tissues examined, only the kidney showed strong and preferential uptake of the substrate. A substantial amount of the administered L-gamma-glutamyl-L-[14C]methionine sulfone was found intact in the kidney; the total uptake of this compound was greater (by about 2-fold) than that of free L-methionine sulfone. Studies with a number of other gamma-glutamyl amino acids and gamma-glutamyl compounds indicate that the kidney has a relatively specific transport system for gamma-glutamyl amino acids. Small but significant amounts of gamma-glutamylmethionine sulfone were found in the liver and pancreas, suggesting that other tissues may also have this system. Transport of gamma-glutamylmethionine sulfone into the kidney was inhibited by inhibitors of glutathione synthesis and of gamma-glutamyl transpeptidase. The results suggest that both the transpeptidase and glutathione may be involved in transport of gamma-glutamyl amino acids.
Identification of transferrin receptors on the surface of human cultured cells.
Authors: Hamilton T A , Wada H G , Sussman H H
Published: May 2006

We have examined the binding of human transferrin to cultured human choriocarcinoma cell lines and to detergent extracts of such cells. The results indicate the presence of a high-affinity saturable binding site (Ka = 4.25 x 10(8) M-1) that is specific for transferrin. This receptor has also been detected on three other human cell lines of different phenotypic origin, including Wil-2 (splenic lymphocytes of B-cell origin), RPMI-2650 (a quasi-diploid nasopharyngeal carcinoma), and WI-38 (embryonic lung fibroblasts). By using anti-human transferrin antiserum to immunoprecipitate the receptor-transferrin complex from detergent extracts of cells containing saturating levels of transferrin followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, a single polypeptide of 90,000 daltons has been identified as a subunit of the putative transferrin receptor. The protein shows immunochemical identity and coelectrophoreses in sodium dodecyl sulfate gels with a cell surface glycoprotein subunit, previously identified in placental brush border membrane preparations, on all human cultured cell lines examined. These results suggest that the recent demonstration of transferrin dependence of maximal cell growth in culture [e.g., Hutchings, S.E. & Sato, G.H. (1978) Proc. Natl. Acad. Sci. USA 75, 901-904] is mediated through expression of this glycoprotein receptor.
Gene for the RNA polymerase sigma subunit mapped in Salmonella typhimurium and Escherichia coli by cloning and deletion.
Authors: Scaife J G , Heilig J S , Rowen L , Calendar R
Published: May 2006

The genes for the RNA polymerase sigma subunit (rpoD) and DNA primase (dnaG) of Salmonella typhimurium have been cloned into lambda vectors. Combined restriction, deletion and functional analysis of the cloned fragment allows us to map the genes precisely on the fragment, establishes the direction in which rpoD is transcribed, and reveals the existence of at least one new gene in the vicinity. A closely homologous, smaller fragment of Escherichia coli DNA, also cloned into lambda, contains rpoD and at least part of dnaG.
Impairment of antigen-presenting cell function by ultraviolet radiation.
Authors: Greene M I , Sy M S , Kripke M , Benacerraf B
Published: May 2006

UV light irradiation of BALB/c mice was found to result in impairment of antigen-presenting cell function. Adherent trinitrophenyl-derivatized cells from the peritoneal exudate cell population or the spleen of UV-treated donors could not induce hapten-specific delayed hypersensitivity responses in UV-irradiated syngeneic mice, whereas adherent trinitrophenyl-derivatized cells from normal mice were able to do so. The failure to induce immunity in UV-treated mice by utilizing UV-treated adherent antigen-presenting cells was associated with the development of antigen-specific suppressor T cells. The implication of these results for UV-induced carcinogenesis is discussed.
Production of choriogonadotropin-like factor by a microorganism.
Authors: Maruo T , Cohen H , Segal S J , Koide S S
Published: May 2006

Extracts from an acetone powder preparation of a culture of a microorganism tentatively named Progenitor cryptocides contain choriogonadotropin (CG)-like factor as determined by radioimmunoassay with antiserum to human (h)CG beta subunit COOH-terminal peptide and radioreceptor assay with bovine corpus luteum membranes. Possible interference by proteases in the extracts was excluded. Immunoreactive materials reacting with antisera to hCG beta subunit and hCG beta subunit COOH-terminal peptide were also found in the extracts. No free alpha subunit was detected. The CG-like factor was purified by chromatography on Sephadex G-100, concanavalin A-Sepharose, and DEAE-Sephadex A-50. The factor was adsorbed by concanavalin A-Sepharose, suggesting that it contains mannose and glucose moieties. The factor was eluted at the same position as standard hCG on Sephadex G-100. It dissociated into two bands when subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis; the bands corresponded to the respective standard hCG subunits. The biological activity of the purified factor as determined by the rat uterine weight assay and the ovarian weight assay was equivalent to 380 (95% confidence limits: 320-490) and 880 (780-1020) international units/mg, respectively. It can be concluded from the present results that a microorganism produces a glycoprotein that is biologically active and has physicochemical properties similar to those of hCG.
Sodium-calcium ion exchange in cardiac membrane vesicles.
Authors: Reeves J P , Sutko J L
Published: May 2006

Membrane vesicles isolated from rabbit ventricular tissue rapidly accumulated Ca2+ when an outwardly directed Na+ gradient was formed across the vesicle membrane. Vesicles loaded internally with K+ showed only 10% of the Ca2+ uptake activity observed with Na+-loaded vesicles. Dissipation of the Na+ gradient with the monovalent cation exchange ionophores nigericin or narasin caused a rapid decline in Ca2+ uptake activity. The Ca2+-ionophore A23187 inhibited Ca2+ uptake by Na+-loaded vesicles and enhanced the rate of Ca2+ loss from the vesicles after uptake. Efflux of preaccumulated Ca2+ from the vesicles was stimulated 30-fold by the presence of 50 mM Na+ in the external medium. Na+-dependent uptake and efflux of Ca2+ were both inhibited by La3+. The results indicate that cardiac membrane vesicles exhibit Na+-Ca2+ exchange activity. Fractionation of the vesicles by density gradient centrifugation revealed a close correspondence between Na+-Ca2+ exchange activity and specific ouabain-binding activity among the various fractions. This relationship suggests that the observed Na+-Ca2+ exchange activity derives from the sarcolemmal membranes within the vesicle preparation.
Adenovirus DNA replication in vitro.
Authors: Challberg M D , Kelly T J
Published: May 2006

A soluble extract from the nuclei of HeLa cells infected with adenovirus 5 (Ad5) carries out the semiconservative replication of exogenously added Ad5 DNA in vitro. Maximal DNA synthesis is observed when DNA-protein complex, isolated from Ad5 virions, is added as template. DNA-protein complex from virions of the closely related virus, adenovirus 2, is also active. In contrast, very little in vitro DNA synthesis is observed when deproteinized Ad5 DNA or DNA from a heterologous source (bacteriophage T7) is added as template. The product of the in vitro reaction consists of long Ad5 DNA strands that are hydrogen-bonded, but not covalently linked, to the input DNA template. During the course of the in vitro reaction, branched molecules with structural features identical to in vivo replication intermediates are formed. These findings support the conclusion that replication in the in vitro system closely resembles adenovirus DNA replication in vivo. The system provides an assay that should be useful for the purification and subsequent characterization of viral and cellular proteins involved in DNA replication.
Temperature-sensitive mutation in the initiation codon of the rIIB gene of bacteriophage T4.
Authors: Belin D , Hedgpeth J , Selzer G B , Epstein R H
Published: May 2006

We have determined the sequence of a ribosome-protected region of T4 rIIB mRNA labeled in vivo. The rIIB mutant HD263, which is temperature sensitive for translation, has an altered initiation codon (AUA instead of AUG). Because wild-type and HD263 proteins have the same fMet peptide, this AUA is used to initiate translation in the mutant rIIB mRNA. We have also identified a six-base sequence which is complementary to the 3" end of the 16S rRNA.
Decreased fidelity of DNA polymerase activity during N-2-fluorenylacetamide hepatocarcinogenesis.
Authors: Chan J Y , Becker F F
Published: May 2006

alpha and beta DNA polymerases (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) were isolated from nuclear and cytoplasmic fractions of rat livers exposed to a carcinogenic regimen with the hepatocarcinogen N-2-fluorenylacetamide and from 24-hr regenerating liver. The fidelity of polymerization of these enzymes was compared by determining the incorporation of noncomplementary deoxyribonucleoside triphosphates (misincorporation) on a poly(dA-dT).poly(dA-dT) template, with MnCl2 and MgCl2 as divalent cations. Our initial studies indicate that the cytoplasmic alpha polymerases from carcinogen-exposed rat livers were strikingly error-prone whereas the nuclear and cytoplasmic beta polymerases retained their fidelity throughout the feeding cycles. The misincorporation was significantly accentuated by MnCl2 compared with that obtained with MgCl2 as divalent cation. The products were sensitive to pancreatic DNase I digestion, indicating that the noncomplementary bases had been incorporated by the polymerization process. Nuclear alpha polymerase showed some degree of infidelity but less than that of cytoplasmic alpha polymerase.
Comparison of primary and secondary stimulation of male rats by estradiol in terms of prolactin synthesis and mRNA accumulation in the pituitary
Authors: Seo Hisao , Refetoff Samuel , Vassart Gilbert , Brocas Huguette
Published: May 2006

Male rats received acute or chronic primary or acute secondary stimulation with estradiol, and the effects on pituitary prolactin synthesis and its mRNA accumulation were examined. Prolactin synthesis was determined by the
in vitro
incorporation of [
3
H]leucine into prolactin over a period of 1 hr. Prolactin mRNA was measured both by cell-free translation in a nuclease-treated rabbit reticulocyte lysate and by hybridization to the complementary DNA. The latter two methods gave similar results under all experimental conditions. Acute primary stimulation with estradiol produced a significant increase in pituitary prolactin mRNA accumulation at 12 hr, which further increased by 2- to 3-fold over the next 48 hr. In contrast, no increase in prolactin synthesis was observed during the first 24 hr. Chronic stimulation with estradiol induced increases of both prolactin synthesis and prolactin mRNA that were quantitatively indistinguishable over the period of 1-4 weeks, reaching a plateau at 5-fold the basal values. By the 13th day after withdrawal of therapy both prolactin synthesis and mRNA had returned to the prestimulation levels. When the effects of estradiol on previously unexposed and estrogen withdrawn animals were compared, it was found that secondary stimulation not only produced a more rapid accumulation of the prolactin mRNA but also abolished the lag period of prolactin synthesis observed during the primary estrogen stimulation. These data demonstrate a lag in the endogenous translation of newly accumulated pituitary prolactin mRNA translatable
in vitro
after primary estrogen stimulation of male rats. The mechanism for the abolition of this lag during the secondary stimulation is now known.
Nucleolar antigen found in several human tumors but not in the nontumor tissues studied.
Authors: Davis F M , Gyorkey F , Busch R K , Busch H
Published: May 2006

Rabbit antibodies to nucleoli isolated from HeLa cells produced bright nucleolar fluorescence in HeLa cells by the indirect immunofluorescence technique. After absorption with fetal bovine serum and placental nuclei, the IgG still produced bright nucleolar fluorescence in 12 human tumor cells including HeLa, HEp-2, cultures of prostate and mammary carcinomas, the Goldenberg GW-39 colon tumor, and biopsy specimens of prostatic, adrenal cortical, thyroid, and squamous cell carcinomas, a hairy cell leukemia of the spleen, a hepatic metastasis of an adenocarcinoma of the colon, and an osteogenic sarcoma. Bright nucleolar fluorescence was not produced in nine nontumor human cells including biopsy specimens of bone marrow, kidney, placenta, thyroid, liver, and prostate, peripheral blood buffy coat, and cultures of normal skin fibroblasts. Nucleolar fluorescence with the absorbed IgG was prevented in HeLa cells by pretreatment of the cells with acid, base, and proteases but not by pretreatment with nucleases; absorption of this IgG with extracts of HeLa nucleoli prevented the nucleolor fluorescence in HeLa and other human tumor cells.