bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet-activating factor (PAF) stimulates the PAF-synthesizing enzyme acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine O2-acetyltransferase and PAF synthesis in neutrophils.
Authors: Doebber T W , Wu M S
Published: May 2006

Platelet activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) induced in isolated rat peritoneal and human peripheral neutrophils a rapid and potent activation of the PAF biosynthetic enzyme acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine O2-acetyltransferase (EC 2.3.1.67). The PAF-induced activation of the neutrophil acetyltransferase (8-10 times basal neutrophil activity) was maximal within 30 sec after PAF addition, as was the PAF-stimulated degranulation. After 1 min of PAF stimulation, the elevated acetyltransferase activity steadily decreased. Within 2 min of stimulation of neutrophils with 10(-6) M PAF, the 7-fold increase in acetyltransferase activity was coincident with substantial PAF synthesis (as measured by [3H]acetate incorporation into PAF), which was 14% of the PAF synthesis induced by the Ca2+ ionophore A23187 at 10(-5) M. PAF activation of the acetyltransferase and PAF synthesis required intact neutrophils as they did not occur in cells broken by sonication. The neutrophil acetyltransferase was 10-30 times more sensitive to activation by PAF than was degranulation as the acetyltransferase activation was evident with 10(-9) M PAF and was about maximal with 3 x 10(-8) M PAF. The unstimulated and PAF-induced acetyltransferase exhibited the same Km for acetyl-CoA (67 microM), but the Vmax for the PAF-induced enzyme (1667 pmol/min per 10(7) cells) was 10 times that of the unstimulated enzyme (175 pmol/min per 10(7) cells). The PAF induction of the acetyltransferase was less sensitive to inhibition by the specific PAF receptor antagonist L-652,731 than was PAF-induced degranulation. This, along with the differing sensitivities to PAF, suggests that acetyltransferase activation and degranulation induced by PAF either involve two different PAF receptors or involve one receptor type with different receptor occupancy requirements. Escherichia coli alkaline phosphatase, which greatly decreased the activity of the acetyltransferase in spleen microsomes, had little or no effect on the basal or PAF-induced neutrophil acetyltransferase. Thus, by stimulating the activity of acetyltransferase, PAF induces in neutrophils the synthesis of more PAF, thereby probably augmenting the neutrophil response to the initial PAF.
Brain-type prostaglandin D synthetase occurs in the rat cochlea.
Authors: Tachibana M , Fex J , Urade Y , Hayaishi O
Published: May 2006

Prostaglandin D synthetase [(5Z, 13E)-(15S)-9 alpha, 11 alpha-epidioxy-15-hydroxyprosta-5,13-dienoate D-isomerase, EC 5.3.99.2] activity was found in the high-speed (100,000 x g, 1 hr) supernatant of the homogenate of the cochlea of adult rats. The specific activity (14.0 nmol/min per mg of protein) was 6- to 7-fold higher than that in the central nervous system. The enzyme showed full activity with 1 mM glutathione, 1 mM 2-mercaptoethanol, or 0.5 mM dithiothreitol and was almost completely inhibited by 1 mM 1-chloro-2,4-dinitrobenzene. The Km value for prostaglandin H2 was about 20 microM. These catalytic properties are the same as those of rat brain prostaglandin D synthetase but different from those of rat spleen prostaglandin D synthetase. The activity decreased to less than 20% of its initial level after incubation with excess amounts of a polyclonal or a monoclonal antibody against the brain enzyme, but the activity remained unchanged with a polyclonal antibody against the spleen enzyme, indicating that the brain-type enzyme synthesizes prostaglandin D2 in the cochlea. When cryosections of 5-week-old (adult) rat cochleas were stained by an immunoperoxidase method with antibodies against the brain enzyme, the immunoreactivity was found in inner and outer hair cells, Claudius' cells, Deiters' cells, marginal cells, basal cells, and cells of Reissner's membrane. In 8-day-old rats, the immunoreactivity was found in all of these cell types except hair cells. The immunoreactivity in hair cells was found in only one specimen from 9-day-old animals, and no immunoreactivity was found in spiral ganglion cells at any of the ages examined. These findings indicate that prostaglandin D2 is produced by the brain-type synthetase in the indicated types of cochlear cells.
Tubulofilaments in negatively stained scrapie-infected brains: relationship to scrapie-associated fibrils.
Authors: Narang H K , Asher D M , Gajdusek D C
Published: May 2006

A simple method was devised for negative-stain transmission electron microscopy of brain infected with the agent of scrapie. Brains of infected hamsters contained large masses of tubulofilamentous structures with irregular fuzzy surfaces. Brains of mice infected with Creutzfeldt-Jakob disease agent contained similar tubulofilaments in smaller numbers. The abnormal tubulofilaments resembled but were distinguished from normal microtubules. On grids soaked in sodium dodecyl sulfate the abnormal tubulofilaments were found in stages of fragmentation, an outer coat appearing t0 strip from the surface to reveal thinner fibrillary structures resembling scrapie-associated fibrils (SAF). The unmasked fibrils were identified as SAF by immunogold labeling, while the larger tubulofilaments were not labeled. The findings indicate that in infected brain tissue SAF may occur as an internal part of a larger structure that is disrupted by detergent and are not likely to be an artifact formed during extraction procedures.
Isolation of temperature-sensitive Abelson virus mutants by site-directed mutagenesis.
Authors: Engelman A , Rosenberg N
Published: May 2006

Mutants of Abelson virus encoding temperature-sensitive protein-tyrosine kinase (EC 2.7.1.112) were created by site-directed mutagenesis using sequence information from temperature-sensitive mutants of the related v-src oncogene. Expression of these two independent mutations in Escherichia coli resulted in reduced phosphorylation of the mutant proteins at high temperature. Viruses containing one of the mutations induced conditional transformation of both NIH 3T3 and lymphoid cells when expressed in the context of a truncated transforming protein. These results underscore the functional homology between protein-tyrosine kinases and suggest that transfer of mutations within a related gene family may provide a rapid method to create mutants.
Epstein-Barr virus (EBV) induces expression of B-cell activation markers on in vitro infection of EBV-negative B-lymphoma cells.
Authors: Calender A , Billaud M , Aubry J P , Banchereau J , Vuillaume M , Lenoir G M
Published: May 2006

A set of B-cell activation markers, including the EBV/C3d receptor [complement receptor type 2 (CR2) (CD21)], the 45-kDa lymphoblastoid cell-associated (Blast-2) antigen (CD23), and the B-cell restricted activation (Bac-1) antigen (which was recently identified as a potential B-cell growth factor receptor) can be turned on by infecting lymphoma cells that are genome negative for Epstein-Barr virus (EBV) with the B95-8 immortalizing strain of the virus. The nonimmortalizing EBV variant, strain P3HR-1, which possesses a deletion within the BamHI WYH region of the genome containing the coding sequence for the EBV-determined nuclear antigen 2, does not induce expression of these markers. Other lymphoblastoid cell-associated antigen markers can be activated by infection with either immortalizing or nonimmortalizing viruses. These results suggest that the immortalizing potential of EBV is correlated with its ability to induce expression of B-cell activation markers, which are suspected to play a major role in the physiological pathway leading to lymphoid cell proliferation. The viral genomic region deleted in the nonimmortalizing strain of EBV seems to be required for activation of some of these markers. Human lymphoma cell lines, such as those used in this study, can thus help identify the specific EBV genes involved in lymphoid B-cell proliferation and the mechanism of action of these genes.
Sequence-directed bent DNA helix is the specific binding site for Crithidia fasciculata nicking enzyme.
Authors: Linial M , Shlomai J
Published: May 2006

The sequence-directed bent structure of kinetoplast DNA minicircles specifies a unique binding site for Crithidia fasciculata nicking enzyme. Binding of the purified enzyme to the bent structure results in the formation of a tight enzyme-DNA complex that is highly specific to curved DNA. Recognition of the binding site is not determined by the nucleotide sequence at the site of binding per se but through the specific local variation in the DNA helix geometry. Both dynamic curved structures, which are generated by supercoiling, and static ones, which are sequenced-directed, could support and efficient enzyme-DNA complex formation. Binding interactions are dependent upon the degree of the helix curvature and decrease with the straightening of the binding site. DNase I protection experiments identify distinct domains of enzyme binding within the bent structure and suggest the induction of structural changes within these regions as a result of protein-DNA interactions.
Specific binding of transposase to terminal inverted repeats of transposable element Tn3.
Authors: Ichikawa H , Ikeda K , Wishart W L , Ohtsubo E
Published: May 2006

Tn3 transposase, which is required for transposition of Tn3, has been purified by a low-ionic-strength-precipitation method. Using a nitrocellulose filter binding assay, we have shown that transposase binds to any restriction fragment. However, binding of the transposase to specific fragments containing the terminal inverted repeat sequences of Tn3 can be demonstrated by treatment of transposase-DNA complexes with heparin, which effectively removes the transposase bound to the other nonspecific fragments at pH 5-6. DNase I "footprinting" analysis showed that the transposase protects an inner 25-base-pair region of the 38-base-pair terminal inverted repeat sequence of Tn3. This protection is not dependent on pH. Interestingly, binding of the transposase to the inverted repeat sequences facilitates DNase I to nick at the end of the Tn3 sequence. It was also observed that the transposase protects the end regions of restriction fragments with a cohesive sequence at their 5' end or with a flush end from DNase I cleavage. The specific and nonspecific binding of transposase to DNA is ATP-independent.
Identification of the alternative terminal oxidase of higher plant mitochondria
Authors: Elthon Thomas E. , McIntosh Lee
Published: May 2006

In addition to cytochrome oxidase, plant mitochondria have a second terminal oxidase called the alternative oxidase. The alternative oxidase is of great interest in that energy is not conserved when electrons flow through it. The potential energy of the system is thus lost as heat, and, in plants with high levels of the alternative oxidase, this results in thermogenesis. We have purified the alternative oxidase from mitochondria of the thermogenic spadix of
Sauromatum guttatum
and have identified its polypeptide constituents by using polyclonal antibodies. A 166-fold purification was achieved through a combination of cation-exchange (carboxymethyl-Sepharose) and hydrophobic-interaction (phenyl-Sepharose) chromatography. Polyclonal antibodies raised to the CM-Sepharose fractions readily immunoprecipitated alternative oxidase activity and immunoprecipitated four of the proteins that copurify with the activity. These proteins have apparent molecular masses of 37, 36, 35.5, and 35 kDa. Polyclonal antibodies raised individually to the 37-, 36-, and 35.5- plus 35-kDa proteins cross-reacted with all of these proteins, indicating the presence of common antigenic sites. The 37-kDa protein appears to be constitutive in
Sauromatum
, whereas expression of the 36- and 35-kDa proteins was correlated with presence of alternative pathway activity. The 35.5-kDa protein appears with loss of alternative pathway activity during senescence, indicating that this protein may be a degradation product of the 36-kDa protein. Binding of anti-36-kDa protein antibodies to total mitochondrial protein blots of five plant species indicated that similar proteins were always present when alternative pathway activity was observed.
Phenotypic properties, interleukin 2 production, and developmental origin of a "mature" subpopulation of Lyt-2- L3T4- mouse thymocytes.
Authors: Ceredig R , Lynch F , Newman P
Published: May 2006

Two-color flow microfluorometry using monoclonal antibodies to cell surface determinants has shown that a subpopulation of mouse Lyt-2- L3T4- thymocytes, comprising 18% of Lyt-2- L3T4- cells in adult C57BL/6 mice, appears in the thymus late during fetal development. These Lyt-2- L3T4- cells are characterized by lack of expression of determinants recognized by monoclonal antibody J11d, a phenotype characteristic of more "mature" functional T cells. This J11d- subpopulation of Lyt-2- L3T4- thymocytes has now been shown to produce significant quantities of interleukin 2 following mitogen stimulation and to express T-cell receptor molecules recognized by monoclonal antibodies KJ-16 and F23.1. Furthermore, culturing of fetal thymus lobes has shown that precursors of this subpopulation of Lyt-2- L3T4- thymocytes are already present in thymus at 14 days of embryonic development and are thus derived from an intrathymic precursor cell. So, within the mouse thymus, phenotypic changes and acquisition of mature T-cell characteristics occur within a subpopulation of cells originally thought of as exclusively "immature."
Isotropic probability measures in infinite-dimensional spaces
Authors: Backus George
Published: May 2006

Every isotropic probability measure on the space
R
∞
of real sequences x = (
x
1
,
x
2
,...) is a convex combination of the measure concentrated at 0 and a member of I
0
(
R
∞
), the set of all isotropic probability measures
p
∞
on
R
∞
with
p
∞
({0}) = 0. Each
p
∞
[unk] I
0
(
R
∞
) is completely determined by any one of its finite-dimensional marginal distributions

p
n

. Each

p
n

has a density function

f
n

with

dp
n

(
x
1
,...,

x
n

) =
dx
1
...

dx
n
f
n

(
x
1
2
+... +

x
n

2
). Each

f
n

is completely monotone in 0 < ξ < ∞ (hence analytic in the right complex ξ half-plane), and π

n
/2

Γ(
n
/2)
-1
ʃ
0
∞
d
ξ ξ

n
/2-1

f
n

(ξ) = 1. Every
f
that satisfies these two conditions is

f
n

for a unique
p
∞
[unk] I
0
(
R
∞
). Hence the equation πʃ
ξ
∞
d
ζ
f
2
(ζ) = ʃ
0
∞
d
μ (
t
)
e

-
t
ξ

defines a bijection between I
0
(
R
∞
) and the set of all probability measures μ on 0 ≤
t
< ∞. If
p
∞
[unk] I
0
(
R
∞
) then
p
∞
({x: Σ

i
=1

∞

x
i

2
< ∞}) = 0, so
p
∞
is not a “softened” or “fuzzy” version of the inequality Σ

i
=1

∞

x
i

2
≤ 1. If the prior information in a linear inverse problem consists of this inequality and nothing else, stochastic inversion and Bayesian inference are both unsuitable inversion techniques.
Clathrin heavy chain: molecular cloning and complete primary structure.
Authors: Kirchhausen T , Harrison S C , Chow E P , Mattaliano R J , Ramachandran K L , Smart J , Brosius J
Published: May 2006

We have deduced the 1675-amino acid sequence of rat clathrin heavy chain from cDNA clones and predict a protein of Mr 191,569. We have established the polarity of the heavy chain and assigned sequence positions to several structural landmarks of the clathrin leg. The terminal domain at the distal end of the clathrin leg is at the amino terminus of the heavy chain. It is connected to the distal segment by a flexible "link" from Tyr-479 to Arg-523. There is an unusual sequence at the carboxyl terminus that may form the globular projection at the vertex of the clathrin trimer. We suggest that a possible site of heavy-chain-light-chain interaction is located in the proximal segment. Comparison with other partially sequenced mammalian clathrin heavy chains shows that the primary structure is highly conserved. The heavy chain is unrelated to other classes of structural proteins.
Detection in situ of genomic regulatory elements in Drosophila.
Authors: O'Kane C J , Gehring W J
Published: May 2006

We have developed an approach for the in situ detection of genomic elements that regulate transcription zin Drosophila melanogaster. The approach is analogous to a powerful method of bacterial genetics, the random generation of operon fusions, that enables the isolation and characterization of genes simply by knowing or postulating their pattern of expression; it is not necessary initially to screen for mutant phenotypes. To apply this approach to Drosophila, we have used the expression of the lacZ gene of Escherichia coli from the P-element promoter in germ-line transformant flies to screen for chromosomal elements that can act at a distance to stimulate expression from this apparently weak promoter. Of 49 transformed fly lines obtained, approximately 70% show some type of spatially regulated expression of the lacZ gene in embryos; many of these express lacZ specifically in the nervous system. The P-lacZ fusion gene is, therefore, an efficient tool for the recovery of elements that may regulate gene expression in Drosophila and for the generation of a wide variety of cell-type-specific markers.
Cloning of the chromosome translocation breakpoint junction of the t(14;19) in chronic lymphocytic leukemia.
Authors: McKeithan T W , Rowley J D , Shows T B , Diaz M O
Published: May 2006

Our laboratory has reported that t(14;19)(q32; q13.1) is a recurring translocation in the neoplastic cells of patients with chronic lymphocytic leukemia. In the present study, we have analyzed the leukemic cells from one such patient with probes from the immunoglobulin heavy-chain locus, which is present on band q32 of chromosome 14. Using a probe for the alpha constant-region gene segments, we detected a rearranged band by Southern blot analysis. This rearranged band was cloned and mapped. A subclone free of repetitive sequences was shown to be from chromosome 19 by analysis of human-mouse somatic cell hybrids, confirming that the rearranged band contains the translocation breakpoint junction. This probe may be used to identify a gene on chromosome 19 adjacent to the breakpoint that can contribute to the malignant development of B lymphocytes.
Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide.
Authors: Ignarro L J , Buga G M , Wood K S , Byrns R E , Chaudhuri G
Published: May 2006

The objective of this study was to determine whether nitric oxide (NO) is responsible for the vascular smooth muscle relaxation elicited by endothelium-derived relaxing factor (EDRF). EDRF is an unstable humoral substance released from artery and vein that mediates the action of endothelium-dependent vasodilators. NO is an unstable endothelium-independent vasodilator that is released from vasodilator drugs such as nitroprusside and glyceryl trinitrate. We have repeatedly observed that the actions of NO on vascular smooth muscle closely resemble those of EDRF. In the present study the vascular effects of EDRF released from perfused bovine intrapulmonary artery and vein were compared with the effects of NO delivered by superfusion over endothelium-denuded arterial and venous strips arranged in a cascade. EDRF was indistinguishable from NO in that both were labile (t1/2 = 3-5 sec), inactivated by pyrogallol or superoxide anion, stabilized by superoxide dismutase, and inhibited by oxyhemoglobin or potassium. Both EDRF and NO produced comparable increases in cyclic GMP accumulation in artery and vein, and this cyclic GMP accumulation was inhibited by pyrogallol, oxyhemoglobin, potassium, and methylene blue. EDRF was identified chemically as NO, or a labile nitroso species, by two procedures. First, like NO, EDRF released from freshly isolated aortic endothelial cells reacted with hemoglobin to yield nitrosylhemoglobin. Second, EDRF and NO each similarly promoted the diazotization of sulfanilic acid and yielded the same reaction product after coupling with N-(1-naphthyl)-ethylenediamine. Thus, EDRF released from artery and vein possesses identical biological and chemical properties as NO.
cDNA cloning and complete sequence of porcine choline acetyltransferase: in vitro translation of the corresponding RNA yields an active protein.
Authors: Berrard S , Brice A , Lottspeich F , Braun A , Barde Y A , Mallet J
Published: May 2006

A cDNA clone encoding the complete sequence of porcine choline acetyltransferase (ChoAcTase; acetyl-CoA: choline O-acetyltransferase, EC 2.3.1.6.) has been identified. A cDNA library, constructed from poly(A)+ RNA of ventral spinal cord, was screened with a mixture of eight oligonucleotides corresponding to the N-terminal sequence of pig brain ChoAcTase. Among five positive clones, one, pChAT-1, was identified as a ChoAcTase cDNA clone based on the following criteria. (i) This clone has an open reading frame coding for a protein of the size expected for ChoAcTase (640 amino acids). (ii) The amino acid composition deduced from the nucleotide sequence of this open reading frame matches that of purified porcine ChoAcTase. (iii) When subcloned in the T7 expression system, the corresponding RNA directs the synthesis in the rabbit reticulocyte lysate of a protein that is specifically immunoprecipitated by antibodies raised against ChoAcTase. (iv) Finally and most important, this corresponding RNA, when translated in the reticulocyte lysate, as well as in the Xenopus oocyte system, directs the synthesis of a protein displaying ChoAcTase activity. This activity is inhibited by the specific ChoAcTase inhibitor 4-(1-naphthylvinyl)pyridine. Comparison of porcine ChoAcTase sequence with that of Drosophila reveals 32% identity between these proteins, when the sequences are suitably aligned. pChAT-1 probe hybridizes with a porcine mRNA species that is at least 7000 nucleotides long, whereas the equivalent rat mRNA species is 3700 nucleotides long.
Expression of cytoskeletal protein 4.1 during avian erythroid cellular maturation.
Authors: Yew N S , Choi H R , Gallarda J L , Engel J D
Published: May 2006

We have isolated a cDNA clone encoding part of protein 4.1, an integral component of the erythrocyte cytoskeleton. The recombinant was isolated by immunological screening of a chicken erythroid lambda gt11 cDNA library using a monoclonal antibody directed against protein 4.1. DNA blot analysis shows that the gene is present as a single copy per haploid chicken genome, while RNA blot analysis reveals the presence of a single mRNA of 7 kilobases in reticulocytes. Message of the same size (in reduced amounts) is also present in an erythroleukemic cell line transformed by avian erythroblastosis virus and is also present in vastly reduced quantities in nonerythroid hemopoietic cells. Immunoblotting and immunofluorescence experiments show that a subset of the chicken 4.1 variant proteins is preferentially expressed during in vitro differentiation of chicken erythroleukemic cells. These data indicate that the gene is both actively transcribed and translated during early erythroid cellular maturation.
Wave energy and intertidal productivity
Authors: Leigh Egbert G. , Paine Robert T. , Quinn James F. , Suchanek Thomas H.
Published: May 2006

In the northeastern Pacific, intertidal zones of the most wave-beaten shores receive more energy from breaking waves than from the sun. Despite severe mortality from winter storms, communities at some wave-beaten sites produce an extraordinary quantity of dry matter per unit area of shore per year. At wave-beaten sites of Tatoosh Island, WA, sea palms,
Postelsia palmaeformis
, can produce > 10 kg of dry matter, or 1.5 × 10
8
J, per m
2
in a good year. Extraordinarily productive organisms such as
Postelsia
are restricted to wave-beaten sites. Intertidal organisms cannot transform wave energy into chemical energy, as photosynthetic plants transform solar energy, nor can intertidal organisms “harness” wave energy. Nonetheless, wave energy enhances the productivity of intertidal organisms. On exposed shores, waves increase the capacity of resident algae to acquire nutrients and use sunlight, augment the competitive ability of productive organisms, and protect intertidal residents by knocking away their enemies or preventing them from feeding.
Modification of erythrocyte enzyme activities by persulfides and methanethiol: possible regulatory role.
Authors: Valentine W N , Toohey J I , Paglia D E , Nakatani M , Brockway R A
Published: May 2006

Sulfhydryl modification of 22 human erythrocyte enzymes was achieved by exposing intact erythrocytes, hemolysates, and partially purified enzymes to persulfides (RSSH) generated nonenzymatically from cystine in the presence of pyridoxal phosphate and mercaptopyruvate, which donates its sulfur to suitable acceptors with the mediation of the carrier enzyme, mercaptopyruvate sulfurtransferase (EC 2.8.1.2). The inhibition pattern was qualitatively similar for persulfides and that previously reported by us for the methylthio-group donor, methyl methanethiosulfonate. Thirteen activities were inhibited, and 9 were minimally or not at all affected. Pyruvate kinase was similarly modified by all systems in terms of phosphoenolpyruvate kinetics, thermostability, and interaction with the negative effector ATP. Partial-to-complete reversal of inhibition was documented in a subset of activities inhibited by mercaptopyruvate upon 30-min incubation with 1 mM dithiothreitol. A possible physiologic role for methylthio groups and for persulfides is discussed.
A specific endpoint assay for ubiquitin.
Authors: Rose I A , Warms J V
Published: May 2006

Simple endpoint assays for free ubiquitin (Ub) and for the Ub-activating enzyme are described. The method for measuring Ub makes use of the reaction of iodoacetamide-treated Ub-activating enzyme (E): [3H]ATP + Ub + E----E X [3H]AMP-Ub + PPi and PPi----2Pi (in the presence of pyrophosphatase). The Ub is then measured by determining the acid-insoluble radioactivity. The reaction is accompanied by a slow enzyme-catalyzed hydrolysis of the complex to AMP plus Ub. The presence of ubiquitin-activating enzyme in excess of Ub by approximately equal to 0.1 microM assures that the steady state will be close to the endpoint for total Ub. A preparation of the activating enzyme from human erythrocytes that does not depend on affinity chromatography is described. Several applications of the assay are presented.
Rapid stimulation by insulin of a serine/threonine kinase in 3T3-L1 adipocytes that phosphorylates microtubule-associated protein 2 in vitro.
Authors: Ray L B , Sturgill T W
Published: May 2006

Insulin treatment (Kact, 5 X 10(-9) M) of serum-starved 3T3-L1 adipocytes stimulates a soluble serine/threonine kinase that catalyzes phosphorylation of microtubule-associated protein 2 (MAP-2) in vitro. Maximal activation of MAP-2 kinase activity by 80 nM insulin was observed after 10 min of hormonal stimulation, prior to maximal stimulation of S6 kinase activity (20 min). The insulin-stimulatable MAP-2 kinase activity is not adsorbed to phosphocellulose, whereas the principal S6 kinase activity is retained and elutes at approximately 0.5 M NaCl. The insulin-stimulatable MAP-2 kinase is less stable during incubation at 30 degrees C than S6 kinase activity. Inclusion of phosphatase inhibitors decreases the rate at which the stimulated MAP-2 kinase activity is lost from extract supernatants incubated at 30 degrees C. p-Nitrophenyl phosphate is more effective than DL-phosphotyrosine, whereas DL-phosphoserine is without effect at the concentration used (40 mM). The difference in MAP-2 kinase activity in extract supernatants from control and insulin-treated cells is also preserved after rapid chromatography on Sephadex G-25. These results show that a soluble serine/threonine kinase is rapidly activated by insulin, possibly by phosphorylation of either the kinase itself or an interacting modulator.
Human salivary gustin is a potent activator of calmodulin-dependent brain phosphodiesterase.
Authors: Law J S , Nelson N , Watanabe K , Henkin R I
Published: May 2006

Human salivary gustin stimulated activity of brain calmodulin-dependent cyclic nucleotide phosphodiesterase (cAMP PDEase; 3',5'-cyclic-nucleotide phosphodiesterase, EC 3.1.4.17) in a dose-dependent manner in the absence of calmodulin. At physiological levels found in human saliva, gustin activated cAMP PDEase 5- to 6-fold. Activation of PDEase occurred with as little as 500 ng of gustin. Comparative sensitivity of activation of PDEase by gustin was intermediate between calmodulin and lysophosphatidylcholine with maximal activation and half-maximal activation (indicated in parentheses) at 3 X 10(-8) M (4.3 X 10(-9) M), 3.4 X 10(-6) M (3.4 X 10(-7) M), and 2.5 X 10(-3) M (4.0 X 10(-5) M) for calmodulin, gustin, and lysophosphatidylcholine, respectively. No other major salivary protein activated PDEase. Anticalmodulin antibody completely inhibited calmodulin-activated cAMP PDEase activity, but the antibody had no effect on gustin-activated cAMP PDEase activity. A sensitive calmodulin RIA indicated that no calmodulin was detected in any gustin preparation that activated cAMP PDEase. Both gustin and calmodulin rendered cAMP PDEase thermally labile to a similar extent and increased Vmax without affecting the apparent Km for the substrate cAMP. Activation by gustin and calmodulin was unaffected by lubrol-PX, trypsin inhibitor, pepstatin A, or leupeptin. In the presence of 1 mM EGTA, gustin activated cAMP PDE 5- to 6-fold, but the activating ability was completely lost after gustin was heated at 100 degrees C for 5 min. In contrast, calmodulin lost all activating ability in the presence of 1 mM EGTA, whereas heating calmodulin at 100 degrees C for 5 min did not affect its activation of cAMP PDEase. Lysophosphatidylcholine-activation of cAMP PDEase, like gustin activation, was unaffected by EGTA, but lysophosphatidylcholine-activation of cAMP PDEase, like calmodulin activation, was unaffected by heating at 100 degrees C for 5 min.
Cytosynalin: a Mr 35,000 cytoskeleton-interacting and calmodulin-binding protein.
Authors: Sobue K , Okabe T , Kadowaki K , Itoh K , Tanaka T , Fujio Y
Published: May 2006

A Mr 35,000 calmodulin-binding protein, which also possesses the ability to interact with a number of cytoskeletal elements, was purified from bovine synaptosomal membranes, using hydroxylapatite and phosphocellulose column chromatography. We call this protein "cytosynalin," which is derived from a combination of the words "cytoskeleton" and "synapsis." Indirect immunofluorescent staining of cytosynalin revealed that it is distributed throughout neural cells including in dendrites, varicosities, and growth cones. In 3T3 cells, cytosynalin is located on the inner surface of the plasma membrane. The staining pattern of cytosynalin observed with anti-cytosynalin antibodies was similar to that observed with antibodies against calspectin (nonerythroid spectrin or fodrin). Cytosynalin binds to calspectin and to tubulin and actin filaments, as determined by binding assays, turbidity measurements, low-shear viscometry, and electron microscopy. Furthermore, after heating at 90 degrees C for 5 min, cytosynalin retains its binding activity toward calmodulin and cytoskeletal elements. These results lead to the conclusion that cytosynalin is colocalized with calspectin at the inner surface of the plasma membrane, probably in association with other cytoskeletal elements.
Evidence for sequential expression of multiple AMP deaminase isoforms during skeletal muscle development.
Authors: Marquetant R , Desai N M , Sabina R L , Holmes E W
Published: May 2006

AMP deaminase (myoadenylate deaminase; EC 3.5.4.6) is an integral part of the myofibril in skeletal muscle, and this enzyme plays an important role in energy metabolism in this tissue. We report here the identification of three AMP deaminase isoforms during skeletal muscle development in the rat. An embryonic isoform is expressed in the developing hindlimb of the rat between 7 and 14 days of gestation. This isoform is not unique to skeletal muscle or the embryo as it is also expressed in many nonmuscle tissues of the perinatal and adult rat. A perinatal isoform of AMP deaminase that is restricted to skeletal muscle is produced 4-6 days before birth and persists for 2-3 weeks of postnatal life. An adult, skeletal muscle-specific isoform of AMP deaminase appears at birth and reaches maximal levels after 3 weeks of postnatal development. We conclude from these studies there is a developmentally controlled program that leads to the sequential expression of AMP deaminase isoforms during the transition from embryonic to adult skeletal muscle.
In vitro modulation of antigen-primed T cells by a glycosylation-inhibiting factor that regulates the formation of antigen-specific suppressive factors.
Authors: Iwata M , Ishizaka K
Published: May 2006

BDF1 [(C57BL/6 X DBA/2)F1] mice were primed with alum-absorbed ovalbumin, and their spleen cells were cultured with ovalbumin to activate antigen-primed T cells. The activated T cells were then propagated in interleukin 2-containing medium in the presence or absence of affinity-purified glycosylation-inhibiting factor (GIF). Upon incubation with ovalbumin-pulsed macrophages, T cells propagated in the absence of GIF produced IgE-potentiating factor and glycosylation-enhancing factor. In contrast, T cells propagated in the presence of GIF produced IgE-suppressive factor and GIF. The ovalbumin-primed T cells propagated in the presence of GIF constitutively produced the 13-kDa GIF that lacked affinity for ovalbumin. However, stimulation of the same T cells with ovalbumin-pulsed macrophages resulted in the production of 80- and 35-kDa GIF that had affinity for ovalbumin. Both the antigen-specific GIF and nonspecific GIF from the activated BDF1 T cells had I-Jb determinants. Since the ovalbumin-specific GIF is derived from Lyt-2+, I-J+ ovalbumin-specific suppressor T cells and suppresses the anti-hapten antibody response to dinitrophenyl-coupled ovalbumin, the results strongly suggest that the presence of GIF during the propagation of antigen-primed T cells facilitates the generation of antigen-specific suppressor T cells.
Environmental changes induced by growth-associated triggering factors in injured optic nerve of adult rabbit.
Authors: Bawnik Y , Harel A , Stein-Izsak C , Schwartz M
Published: May 2006

Central nervous system (CNS) neurons of mammals regenerate poorly after axonal injury. However, if an injured CNS neuron (rabbit optic nerve) is supplied with appropriate soluble substances ("growth-associated triggering factors") derived from medium conditioned by regenerating fish optic nerve or newborn rabbit optic nerve, it can express regeneration-associated characteristics. Such characteristics include a general increase in protein synthesis, changes in synthesis of specific polypeptides, and sprouting of nerve fibers in culture. The present study of rabbit optic nerves demonstrates that such active substances affect the neuronal environment (i.e., the non-neuronal cells), thereby perhaps causing a shift in the environment from an inhibitory to a regenerative supportive one. Apparently, such an environment is spontaneously achieved in injured CNS nerves of lower vertebrates (e.g., fish optic nerves), which are regenerable. Treatment of injured rabbit optic nerve with soluble factors from medium conditioned by regenerating carp optic nerve resulted in a selective increase in proliferation ([3H]thymidine incorporation) of perineural cells and the appearance of a 12-kDa polypeptide in a homogenate derived from the nerve and its associated cells. This polypeptide may be related to growth, since it comigrates in NaDodSO4/polyacrylamide gel electrophoresis with a 12-kDa polypeptide that is continuously present in a regenerative system. In addition, there were injury-induced changes in the polypeptides of the nerve that were independent of treatment with conditioned medium and were correlated with nerve maturation. The most prominent changes of this type were in 18-kDa and 25-kDa polypeptides whose levels were reduced after injury and were found to be correlated with the nerve maturation (myelination) state.
The lethal yellow allele-associated provirus results in the production of chimeric viral-host RNAs.
Authors: Lovett M , Epstein C J
Published: May 2006

The lethal yellow (Ay) mutation is the most dominant allele at the agouti locus of the mouse. Mice heterozygous for this allele have a yellow coat color, are genetically obese with an increased susceptibility to cancer, and have other metabolic derangements. Mice homozygous for Ay die early in embryogenesis, possibly because of a trophectoderm defect. The Ay mutation is distinguished from the many alleles described at the agouti locus in being associated with an endogenous provirus, designated endogenous ecotropic murine leukemia viral locus 15 (Emv-15). To obtain DNA sequences from regions close to or within the agouti locus, we have isolated the Emv-15 provirus and have found that the DNA sequences adjacent to the provirus are part of a mouse gene that is expressed in the same transcriptional orientation as the proviral genes. These mouse DNA sequences recognize two distinct size classes of RNA in various adult wild-type tissues including skin. In Ay heterozygotes both types of transcript hybridize to the proviral long terminal repeat, and, in heterozygous spleens, the shorter transcript is present at an enhanced steady-state level. These results suggest that the Ay-associated provirus (Emv-15) is distinguished from the endogenous C-type proviruses described to date in having altered the expression of a flanking host gene by promoter insertion, resulting in the production of chimeric viral-host fusion transcripts.
On-line visualization of dendritic release of acetylcholinesterase from mammalian substantia nigra neurons.
Authors: Llinás R R , Greenfield S A
Published: May 2006

This study presents, to our knowledge, the first on-line measurement of acetylcholinesterase (AcChoEase) release from brain tissue. It is now well established that a soluble form of the enzyme is released from central nervous system neurons, and it has been proposed on indirect grounds that such release may occur not only from presynaptic terminals but also from the dendrites of dopamine-containing nigrostriatal neurons. We have used a chemiluminescent reaction to examine the real-time release of AcChoEase from the substantia nigra in vitro in brainstem slices. The light emission was captured by two fiber optic systems, one in direct contact with the brain slice from below and the second 4-mm above the slice, allowing simultaneous imaging of the emitted light and quantitative photometry. It was determined that the light signals are not due to the spontaneous hydrolysis of acetylcholine or the presence of free choline, but are caused by the enzymatic action of AcChoEase. Using this technique, it can be directly shown that AcChoEase is spontaneously released from the soma or dendrites of nigral neurons. The release of the enzyme, which is stored in the subcisternal dendritic compartment, is resistant to blockade of voltage-dependent sodium conductances, is calcium dependent, and can be increased by addition of potassium to the bathing solution. The procedure we describe here will make it possible to study the release of endogenous AcChoEase on a time-scale close to that over which it functions.
Native genomic blotting: high-resolution mapping of DNase I-hypersensitive sites and protein-DNA interactions.
Authors: Pauli U , Chrysogelos S , Stein J , Stein G
Published: May 2006

DNase I-hypersensitive sites are observed in the promoter regions of actively expressed genes, potentially active genes, and genes that were once active. We have developed an approach that greatly increases the resolution for mapping these sites by electrophoresing genomic DNA on native polyacrylamide gels prior to electroblotting and hybridization. This improved method has been used to scan the promoter and coding region of a cell-cycle-dependent human histone H4 gene with an accuracy of +/-5-10 base pairs. Protein-DNA interactions can be seen in the autoradiograph as light areas and DNase I-hypersensitive sites as dark bands. Therefore, this method provides a rapid and relatively simple means to accurately localize protein-DNA interactions as well as DNase I-hypersensitive sites, thus directly displaying DNase I hypersensitivity and protein-DNA complexes on one autoradiograph. It also potentially allows the analysis of small changes in DNase I-hypersensitive sites under various biological conditions. With this technique rather large regions of DNA can be screened to determine areas that should be analyzed by more sophisticated methods, such as genomic sequencing or gel retardation assays.
Nickel-deficient carbon monoxide dehydrogenase from Rhodospirillum rubrum: in vivo and in vitro activation by exogenous nickel.
Authors: Bonam D , McKenna M C , Stephens P J , Ludden P W
Published: May 2006

An inactive, Ni-deficient form of carbon monoxide (CO) dehydrogenase [carbon-monoxide:(acceptor) oxidoreductase; EC 1.2.99.2], designated apo-CO dehydrogenase, accumulated in Rhodospirillum rubrum when cells were grown in the absence of Ni and treated with CO. In vivo, both CO dehydrogenase activity and hydrogenase activity increased several hundred fold upon addition of 2 microM NiCl2. Apo-CO dehydrogenase was purified to homogeneity and differed from holo-CO dehydrogenase only in its activity and Ni content, containing less than 0.2 mol of Ni per mol of protein, and a specific activity of 35 mumol of CO per min per mg. Optimal in vitro activation of purified apo-CO dehydrogenase resulted in an enzyme with a specific activity of 2640 mumol of CO per min per mg. No additional enzymes or low molecular weight cofactors were required for activation. Apo-CO dehydrogenase was not activated by MgCl2, MnCl2, CuCl2, ZnCl2, CoCl2, or Na2MoO4. 63Ni was incorporated into apo-CO dehydrogenase during activation. The electron paramagnetic resonance (EPR) spectra of dithionite-reduced apo- and holo-enzyme were identical, indicating that, in the reduced state, the Fe-S centers observed by EPR are unchanged in the apo-enzyme.
Structural alteration in the MYB protooncogene and deletion within the gene encoding alpha-type protein kinase C in human melanoma cell lines.
Authors: Linnenbach A J , Huebner K , Reddy E P , Herlyn M , Parmiter A H , Nowell P C , Koprowski H
Published: May 2006

A correlative study was done to determine possible relationships between nonrandom aberrations in chromosomes 1, 6, and 7 occurring in human cutaneous malignant melanoma and the structure of oncogenes as well as specific genes encoding growth factors and growth factor receptors. Thirty cell lines derived from primary or metastatic melanomas of 28 patients were analyzed by Southern blotting with nick-translated probes for 28 different genes, some of which map near frequent chromosomal breakpoints observed in melanoma. An alteration in the MYB protooncogene was observed in a cell line derived from a primary melanoma in the vertical growth phase, which correlated with a 6q22 chromosomal abnormality. Another primary melanoma cell line had a cytogenetically undetected tumor-specific deletion within the gene for alpha-type protein kinase C. Polymorphic alleles for the genes encoding the epidermal growth factor receptor and alpha-type protein kinase C were also observed.
Douglas' solution of the Plateau problem
Authors: Guillemin Victor , Kostant Bertram , Sternberg Shlomo
Published: May 2006

Using ideas suggested by some recent developments in string theory, we give here an elementary demonstration of one of the key steps in Douglas' celebrated proof of the existence of solutions of the Plateau problem in
n
dimensions.
Prognostic significance of "short-term" effects of chemotherapy on MYC and histone H3 mRNA levels in acute leukemia patients.
Authors: Venturelli D , Lange B , Narni F , Selleri L , Mariano M T , Torelli U , Gewirtz A M , Calabretta B
Published: May 2006

We have found that administration of chemotherapy alters expression of growth-regulated genes in leukemia blast cells. To determine if such changes might be correlated with therapeutic outcome, we studied steady-state mRNA levels of MYC and histone H3 in the leukemic blasts of patients just prior to and 24 hr after the administration of the first doses of antileukemic drug therapy. Among nine patients with acute myelogenous leukemia, mRNA levels of MYC and histone H3 were reduced in five patients, and hematologic remission was achieved in three of these individuals. No remission was obtained in the four patients without reduction in MYC and histone H3 mRNA. Among acute lymphocytic leukemia patients, the mRNA levels of MYC and/or histone H3 were reduced by the therapy in seven of nine patients. A complete hematologic remission was obtained in five of them, and a partial remission was obtained in the other two. No remission was obtained in the patients in which MYC and H3 mRNA levels were unaffected by the therapy. These studies are of interest because they suggest that a decrease in the mRNA levels of MYC and histone H3 24 hr after a single dose of antineoplastic drugs may predict which patients will achieve complete remission; lack of reduction in these mRNAs correlates with failure to achieve remission. In addition, these studies also provide further proof of the heterogeneity of altered growth regulation among human leukemias.
Single K+ channels activated by D2 dopamine receptors in acutely dissociated neurons from rat corpus striatum.
Authors: Freedman J E , Weight F F
Published: May 2006

Corpus striatum neurons acutely dissociated from the brains of young adult rats had membrane surfaces suitable for G omega-seal recording. Whole-cell current-clamp and voltage-clamp recordings indicated that the cells remained electrically excitable after dissociation. Cell-attached recordings frequently revealed single-channel openings in the presence of dopamine or of the D2 dopamine agonist quinpirole. Channel openings were rarely or never observed in the absence of drugs or in the presence of quinpirole plus the dopamine antagonist haloperidol. The D2 antagonist spiperone was more potent at blocking the appearance of the channel than was the D1 antagonist SCH-23390. The channel reversal potential varied with the extracellular K+ concentration as predicted by the Nernst equation. The channel current-voltage relationship was linear, with a conductance of approximately equal to 85 pS in the presence of 140 mM KCl. These results are consistent with the opening of single K+ channels following D2 dopamine receptor activation.
Complex transcription of the Epstein-Barr virus BamHI fragment H rightward open reading frame 1 (BHRF1) in latently and lytically infected B lymphocytes.
Authors: Austin P J , Flemington E , Yandava C N , Strominger J L , Speck S H
Published: May 2006

Several cDNA clones containing the Epstein-Barr virus BamHI fragment H rightward open reading frame 1 (BHRF1) have been recovered from the tightly latent lymphoblastoid cell line IB4. These clones contain the 5' leader exons encoded in the major internal repeat 1 and the viral BamHI fragment Y, identified in the rightwardly transcribed viral mRNAs associated with the latent viral life cycle. In addition, a cDNA clone containing BHRF1 from the Burkitt lymphoma cell line Jijoye was also recovered and exhibits a distinctive splicing pattern. In vitro transcription and translation of BHRF1, followed by immunoprecipitation with Epstein-Barr virus-positive human sera, indicates that this viral antigen is expressed during infection. RNA blot analyses with a wide panel of lymphoblastoid and Burkitt lymphoma cell lines revealed a complex pattern of transcription. Hybridization data obtained with several probes is presented.