bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of growth of the transplantable rat chondrosarcoma by analogs of hypothalamic hormones
Authors: Redding T. W. , Schally A. V.
Published: May 2006

The Swarm chondrosarcoma is a hormone-responsive tumor whose growth is dependent on growth hormone, somatomedins, and glucocorticoids. Our previous work showed that partial functional hypophysectomy can be achieved by chronic administration of the luteinizing hormone-releasing hormone (LH-RH) analog [D-Trp
6
]LH-RH, which lowers blood levels of LH and follicle-stimulating hormone. We have also demonstrated that somatostatin (SS)-28 or analogs of SS-14 depress serum prolactin, growth hormone, and corticotropin (ACTH) levels. Consequently, we investigated the effect of subcutaneous injection of these analogs on the growth of Swarm chondrosarcoma 3 days after transplanting it into male Sprague-Dawley rats. At autopsy, tumor volume was measured and tumors and various organs were weighed. In rats treated with three different analogs of SS-14, [
p
-NH
2
-Phe
4
]SS, [D-5-F-Trp
8
]SS, and [D-5-MeO-Trp
8
]SS, in doses of 30 μg once or twice daily for 14-30 days, there was a significant reduction in tumor volume and/or weight as compared with control rats. The longer acting SS-28 or its analog Val-Gly-Tyr-Val-Ile-Leu-Gly-SS-28, given in doses of 30 μg/day for 22-30 days, also significantly decreased tumor weight and/or volume. In three experiments, [D-Trp
6
]LH-RH (30-60 μg/day), administered alone or together with analogs of SS-14, decreased tumor weight and/or volume. Serum growth hormone and prolactin levels in rats bearing the tumors were significantly decreased after treatment with [D-5-F-Trp
8
]SS or with [D-Trp
6
]LH-RH. The inhibition of growth of the Swarm chondrosarcoma in rats by these analogs suggests that they might lead to a new endocrine therapy for chondrosarcomas, osteosarcomas, and related hormone-dependent neoplasias.
DNA-mediated alteration of the reversion frequency of transformed NIH/3T3 cells.
Authors: Baker R F
Published: May 2006

Cell selection immediately after DNA-mediated transfection of whole-cell DNA into mammalian cells has been used to select for specific DNA sequences that cause a phenotypic effect. Whole-cell mouse or human DNA was cleaved into a distribution of lengths (0.4-25 kilobase pairs) and transfected into anchorage-independent spontaneously transformed NIH/3T3 cells. Immediately after transaction, anchorage-dependent serum concentration-dependent reverents were selected. The Hirt supernatant, containing extrachromosomal DNA resulting from the transfection, was isolated from the revertants and transfected with high molecular weight carrier DNA into a second population of transformed cells; revertants were again selected. After five to seven cycles of transfection of Hirt supernatant DNA (obtained from revertants selected at the previous cycle) into new populations of transformed cells at each cycle, the reversion frequency had become 5-15 times greater than the spontaneous reversion frequency measured for several subclones of nontransfected or mocktransfected transformed NIH/3T3 cells. When nonmammalian genomic DNAs were used in transfecting a first population of cells, there was no effect on the reversion of frequency even after six cycles of selection. The reversion-enhancing activity of sixth-cycle Hirt supernatant DNA resulting after transfection at the first cycle with mouse or human sequences was destroyed by EcoRI but not by BamHI or Sal I. Sequences resembling human Alu I sequences were found in mouse whole-cell DNA isolated from sixth-cycle revertants generated after transfection of human sequences at the first cycle.
Stability of erythrocyte ghosts: a gamma-ray perturbed angular correlation study.
Authors: Kruse C A , Tin G W , Baldeschwieler J D
Published: May 2006

The structural integrity of erythrocyte ghosts made by the preswell and slow-dialysis techniques has been studied in vitro by use of gamma-ray perturbed angular correlation (PAC) techniques and also by standard in vitro leakage methods employing sequestered labeled markers. Complexes of 111In3+ and nitrilotriacetate were encapsulated in ghosts made from human, rabbit, rat, and mouse erythrocytes, and their leakage was monitored by both methods. In addition, 125I-labeled bovine serum albumin was encapsulated, and ghost integrity was monitored by conventional leakage measurements. With the PAC technique the percentage of material released from human ghosts was determined quantitatively, and the results were equivalent to those obtained by the conventional method. In addition, at various times after intravenous injection, tissue distribution of the ghosts in the mouse was studied. The percent injected dose per gram of tissue of the labeled surface proteins of erythrocyte ghosts in circulation approximated that of the entrapped labeled albumin. This suggests that the ghost membrane and contents are strongly associated in vivo. Large 125I-labeled bovine serum albumin molecules and small 111In3+-nitrilotriacetate complexes were delivered in high quantitites to the lung initially, and to the liver and spleen. Because erythrocyte ghosts have the ability to entrap a wide range of substances and deliver them to specific organs, ghosts may be preferable to other drug carriers or drug therapy for treatment of certain disorders.
Promoter-proximal pausing by RNA polymerase II in vitro: transcripts shorter than 20 nucleotides are not capped.
Authors: Coppola J A , Field A S , Luse D S
Published: May 2006

We have synthesized RNA from cloned adenovirus 2 late promoter DNA in an in vitro transcription extract under UTP-limiting conditions. Under these circumstances, almost all of the alpha-amanitin-sensitive RNA produced is shorter than 20 nucleotides; most of these short transcripts are present in four species, 6, 7, 13, and 17 nucleotides. These short RNAs are initiated at the adenovirus 2 promoter, as judged by partial sequence analysis and by the abolition of their synthesis upon cleavage of the template DNA at sites which also abolish the production of full-length transcripts. All of the short transcripts can be chased, with excess UTP, into 197-base run-off transcripts; thus, these RNAs are precursors of full-length transcripts and not synthetic "dead ends." Significantly, none of these short RNAs is capped or 2'-O-methylated. However, 79-base run-off transcripts synthesized from this promoter with nonlimiting NTP levels are fully capped.
Clathrin-coated vesicles contain an ATP-dependent proton pump.
Authors: Forgac M , Cantley L , Wiedenmann B , Altstiel L , Branton D
Published: May 2006

Clathrin-coated vesicles isolated from calf brain contain an ATP-dependent proton pump. Proton movement was monitored by measuring [14C]methylamine distribution. Addition of Mg2+ and ATP to coated vesicles equilibrated with [14C]methylamine resulted in the generation of a 4- to 5-fold concentration gradient, corresponding to a delta pH of 0.6-0.7 units between the medium and the acidic inside of the coated vesicles. ATP-dependent [14C]methylamine uptake was abolished by the proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and partially inhibited by the carboxyl reagent N,N'-dicyclohexylcarbodiimide but was unaffected by the Na+, K+-ATPase inhibitors strophanthidin (100 microM) and vanadate (10 microM) and the mitochondrial ATPase inhibitors oligomycin (10 microgram/ml) and aurovertin (1 microgram/ml). GTP, but not the nonhydrolyzable analog 5'-adenylyl imidodiphosphate, could support [14C]methylamine uptake. Dissipation of the membrane potential with K+ and valinomycin resulted in stimulation of [14C]methylamine uptake, whereas both FCCP and valinomycin stimulated the strophanthidin-resistant ATPase activity. These results are consistent with the existence of an electrogenic, ATP-dependent proton pump in clathrin-coated vesicles. This proton pump may play a role in the acidification events that are essential in receptor-mediated endocytosis.
Identification of adenylate cyclase-stimulating activity and cytochemical glucose-6-phosphate dehydrogenase-stimulating activity in extracts of tumors from patients with humoral hypercalcemia of malignancy.
Authors: Stewart A F , Insogna K L , Goltzman D , Broadus A E
Published: May 2006

Humoral hypercalcemia of malignancy (HHM) most commonly results from secretion by tumors of an unidentified circulating calcemic factor that appears in clinical studies to stimulate both a parathyroid hormone (PTH)-sensitive proximal tubular adenylate cyclase and a distinct PTH-sensitive renal tubular glucose-6-phosphate dehydrogenase complex. In the present study, 8 M urea extracts of tumors from patients with HHM have been shown to contain both in vitro adenylate cyclase-stimulating activity and glucose-6-phosphate dehydrogenase-stimulating activity as detected in a sensitive cytochemical bioassay. Both the adenylate cyclase-stimulating activity and cytochemical bioactivity are due to specific binding of a substance in the tumor extracts to renal PTH receptors, as demonstrated by competitive inhibition studies using the bovine PTH fragment analogue [Nle8,18, Tyr34]bPTH-(3-34) amide. Preincubation with an antiserum to PTH results in no loss of activity in the tumor extract, and the activity appears both on gel filtration and ultrafiltration to be far larger than PTH (estimated Mr 70,000). These studies demonstrate that extracts of tumors from patients with HHM contain a substance that binds to the PTH receptors in the nephron responsible for activation of both the PTH-sensitive glucose-6-phosphate dehydrogenase and the PTH-sensitive adenylate cyclase. This substance is chromatographically and immunologically distinct from PTH. Its role in the genesis of HHM requires further study.
Inactivation of key metabolic enzymes by mixed-function oxidation reactions: Possible implication in protein turnover and ageing
Authors: Fucci Laura , Oliver Cynthia N. , Coon Minor J. , Stadtman Earl R.
Published: May 2006

Several mixed-function oxidation systems catalyze the inactivation of
Escherichia coli
glutamine synthetase. Inactivation involves modification of a single histidine residue in each enzyme subunit and makes the enzyme susceptible to proteolytic degradation. We show here that 10 key enzymes in metabolism are inactivated by a bacterial NADH oxidase and by an oxidase system comprised of NADPH, cytochrome P-450 reductase, and cytochrome P-450 isozyme 2 from rabbit liver microsomes. Most of the inactivatable enzymes require a divalent cation for activity and all but one (enolase) possess a nucleotide binding site. Glutamine synthetase, pyruvate kinase, and phosphoglycerate kinase are protected from inactivation by their substrates; substrate protection of other enzymes was not tested. We propose that inactivation involves mixed-function oxidization system-catalyzed synthesis of H
2
O
2
and reduction of Fe(III) to Fe(II) followed by oxidation of enzyme-bound Fe(II) by H
2
O
2
to generate oxygen radicals that attack a histidine (or other oxidizable amino acid) at the metal binding site of the enzyme. This is supported by the following: (
i
) most of the inactivation reactions are inhibited by EDTA and by catalase, (
ii
) both mixed-function oxidation systems reduce Fe(III), and (
iii
) H
2
O
2
together with Fe(II) catalyzes nonenzymic inactivation of glutamine synthetase. In view of the fact that inactivation of glutamine synthetase makes it susceptible to proteolytic degradation, it is possible that mixed-function oxidation system-catalyzed inactivation of enzymes is a regulatory step in enzyme turn-over. In addition, the implication of oxidative inactivation reactions in ageing is suggested by the fact that many of the enzymes inactivated by mixed-function oxidation systems are known to accumulate as inactive forms during ageing.
Evidence for an altered adenovirus DNA polymerase in cells infected with the mutant H5ts149
Authors: Friefeld Beth R. , Lichy Jack H. , Hurwitz Jerard , Horwitz Marshall S.
Published: May 2006

The N complementation group of adenovirus (Ad) serotype 5 mutants, which are temperature sensitive for viral DNA synthesis
in vivo
, has been used to study a 140,000-dalton DNA polymerase (Pol) that copurified with the 80,000-dalton terminal protein precursor (pTP). Extracts prepared from HeLa cells infected with the N group mutant H5ts149 at nonpermissive temperature were unable to synthesize viral DNA. The defect in these extracts was specifically reversed by addition of the Pol purified from wild-type Ad-infected cytosol. Addition of the pTP, free of the Pol, did not restore replicative activity to H5ts149 extracts. The reactions studied depend on the presence of the DNA template and include the initiation reaction (the covalent attachment of dCMP to the pTP) and the selective replication of Ad DNA restriction endonuclease fragments containing the origin sequences. Glycerol gradient sedimentation showed that a replicative activity representing the pTP-Pol complex was greatly reduced in H5ts149 extracts as compared with wild-type extracts, suggesting some alteration in the mutant. A pool of pTP free of Pol was detected on these gradients in extracts from both wild-type and H5ts149-infected cells. In addition, the initiation and elongation of Ad DNA catalyzed by H5ts149 extracts prepared from cells grown at permissive temperatures was more labile to urea inactivation than extracts prepared from cells infected with wild-type virus. These results, considered together with the mapping of the H5ts149 mutation within an open reading frame approximately large enough to code for the 140,000-dalton DNA polymerase [Gingeras, T. R., Sciaky, D., Gelinas, R. E., Bing-Dong, J., Yen, C. E., Kelly, M. M., Bullock, P. A., Parsons, B. L., O'Neill, K. E. & Roberts, R. J. (1982)
J. Biol. Chem.
257, 13475-13491; Alestrom, P., Akusjarui, G., Pettersson, M. & Pettersson, U. (1982)
J. Biol. Chem.
257, 13492-13498], suggest that the Pol is a virally encoded protein, as is the pTP.
Mouse macrophage Fc receptor for IgG gamma 2b/gamma 1 in artificial and plasma membrane vesicles functions as a ligand-dependent ionophore.
Authors: Young J D , Unkeless J C , Kaback H R , Cohn Z A
Published: May 2006

We tested the effect of specific ligands to the mouse macrophage IgG gamma 2b/gamma 1 Fc fragment receptor (FcR) on ion permeability of plasma membrane vesicles prepared from J774 macrophages by nitrogen cavitation. The monoclonal antibody directed against IgG gamma 2b/gamma 1 FcR (gamma 2b/gamma 1 FcR), 2.4G2 IgG, and soluble and immobilized immunocomplexes induces a dramatic cation flow through plasma membrane vesicles, as measured by [3H]tetraphenylphosphonium+ accumulation. Challenge with the monovalent 2.4G2 Fab also produces an ion flow but the effect is smaller by a factor of 2, and three other monoclonal antibodies directed against major surface antigens of mouse macrophages produce no net ion flow. Membrane vesicles incubated with FcR ligands do not discriminate between Na+ and K+ and show low permeability to Ca2+. gamma 2b/gamma 1 FcR was purified by using monoclonal 2.4G2 and reconstituted into proteoliposomes. Under these circumstances, the purified receptor increased the cation permeability of the proteoliposomes in the presence of specific ligands. The data indicate that the gamma 2b/gamma 1 FcR of J774 macrophages functions as a ligand-dependent ionophore. The ion influx into macrophages mediated by the FcR may play an important role as a signal for internalization of membranes and stimulus-secretion coupling.
Evolution of hybrid dysgenesis determinants in
Drosophila melanogaster
Authors: Kidwell Margaret G.
Published: May 2006

Hybrid dysgenesis is manifested as a group of correlated aberrant genetic traits such as sterility, increased mutation rate, and male recombination. Previous work has shown that it appears when males of strains carrying either of two independent families of transposable elements called
I
and
P
factors are hybridized with females of susceptible strains called
R
and
M
, respectively. Here the results of an extensive survey for dysgenic potential in
Drosophila melanogaster
strains are reported. Striking temporal trends in the distribution of strains were observed with respect to the two transposable element systems; in particular, the frequency of
R
and
M
strains is positively correlated with laboratory age. In recent tests of strain samples, those collected from nature about 50 years ago were the earliest observed to possess
I
characteristics. The
I
type was increasingly frequent in samples from strains more recently originating in the wild. This type is apparently ubiquitous in present day natural populations. the
P
type was not found in strain samples collected before 1950, and collections made subsequently showed increasing frequencies of
P
-factor activity with decreasing laboratory age. Marked geographical patterns are documented in the contemporary worldwide distribution of variant strains within the
P
-
M
system.
M
strains are currently fairly common in natural populations from various parts of the world, except on the American continent where they are rare. The degree and distribution of quantitative variation within
M
and
P
strain categories is related to their time of origin in the wild. The implications of these results are discussed in relation to the hypothesis that hybrid dysgenesis determinants have evolved recently in natural populations and to an alternative hypothesis of laboratory evolution.
Corticotropin-releasing factor (CRF)-like immunoreactivity in the vertebrate endocrine pancreas.
Authors: Petrusz P , Merchenthaler I , Maderdrut J L , Vigh S , Schally A V
Published: May 2006

The light microscopic immunocytochemical localization of corticotropin-releasing factor (CRF) is described in the endocrine pancreas of several species representing the major classes of vertebrates: fishes (channel catfish, Ictalurus punctatus), amphibians (African clawed toad, Xenopus laevis), reptiles (chameleon, Anolis carolinensis), birds (chicken, Gallus domesticus), and several mammals (rat, mouse, cat, rhesus monkey, and man). The CRF-containing cells are scattered over the entire islet tissue in primates and cat, whereas in rat and mouse they are located at the periphery of the islets. In the chicken and catfish, the CRF-containing cells are found in a central location within islets and form larger clusters or cords. Single cells with CRF-like immunoreactivity are interspersed between acinar cells of the exocrine pancreas in all species studied. The CRF cells show a substantial topographical overlap with glucagon cells, but their precise identity and function remain to be determined.
Involvement of kinases in glucose and fructose uptake by Saccharomyces cerevisiae.
Authors: Bisson L F , Fraenkel D G
Published: May 2006

Uptake of glucose, fructose, and the nonmetabolizable analog 6-deoxyglucose was measured in wild-type Saccharomyces cerevisiae and two mutant strains, one (hxk1 hxk2) lacking both hexokinase A(P-I) and B(P-II) but containing glucokinase (and hence able to grow on glucose but not fructose) and the other (hxk1 hxk2 glk) also lacking glucokinase (and not able to grow on glucose either). Uptake of the nonmetabolized substances (i.e., 6-deoxyglucose in all three strains, fructose in the two mutants, and glucose in the triple mutant) reached a plateau at or below the external concentration. The kinetic characteristics of uptake were determined from 5-sec incubations by plotting velocity (V) vs. velocity/substrate concentration (V/S) curves. According to such plots, in the wild-type strain uptake had two components, "high affinity uptake" with Km values of ca. 1 mM for glucose and 6 mM for fructose and "low affinity uptake" with Km values of ca. 20 and 50 mM, respectively. The double kinase mutant showed both components for glucose but only the high Km component for fructose, while the triple kinase mutant showed only high Km uptake for both glucose and fructose. Genetic analysis showed that only in strains lacking both hexokinases (hxk1 hxk2) was the low Km system for fructose absent. Low Km uptake was restored to the triple mutant by introduction of the cloned wild-type genes: HXK1 or HXK2, for fructose uptake, and HXK1, HXK2, or GLK1, for glucose uptake. A phosphoglucose isomerase mutant had both low and high Km uptake for glucose. These results indicate the presence of two types of uptake mechanism for glucose and fructose in yeast, the functioning of one of which, the low Km system, is influenced by the cognate kinases.
Voltage-dependent calcium channel in the squid axon.
Authors: DiPolo R , Caputo C , Bezanilla F
Published: May 2006

A voltage-dependent inward calcium current insensitive to tetrodotoxin has been measured in internally perfused or dialyzed squid giant axons. Sodium conductance was blocked by tetrodotoxin, and potassium conductance was irreversibly destroyed by the lack of potassium in the external and internal medium. Chloride conductance was eliminated by replacement of chloride with methylsulfonate. The calcium current was activated at about -40 mV and it peaked at about 0 mV. The peak inward current was 3 microA/cm2 when external Ca was 80 mM and 0.9 microA/cm2 in 8 mM Ca. In 80 mM Ca, the calcium current is turned on in less than 10 msec, and it does not decay appreciably for pulses up to 70 msec in duration. Barium can replace calcium, and cadmium blocks the calcium current.
Do tissues other than the kidney produce 1,25-dihydroxyvitamin D3 in vivo? A reexamination.
Authors: Shultz T D , Fox J , Heath H , Kumar R
Published: May 2006

Recent experiments have shown that 1,25-dihydroxyvitamin D3-like material is produced in cultured nonrenal cells and may be present in the sera of anephric patients. We reexamined the question of whether 1,25-dihydroxyvitamin D3 can be synthesized extrarenally in the rat in vivo. To intact, sham-operated, ureter-ligated, or acutely nephrectomized vitamin D-deficient rats raised on a diet normal in calcium and phosphorus, we gave a physiologic dose of high-specific-activity 25-hydroxy-[3H]vitamin D3 (3.6-3.8 microCi; approximately equal to 25 pmol per rat). Twenty-four hours later we examined their tissues and plasma for the presence of radiolabeled 1,25-dihydroxyvitamin D3. Large amounts of radioactivity that behaved chromatographically as identical with authentic 1,25-dihydroxyvitamin D3 were present in the plasma, bone, and intestine of the intact, sham-operated, or ureter-ligated rats. However, no radioactivity eluting in a manner similar to 1,25-dihydroxyvitamin D3 was found in plasma, bone, or intestine of acutely nephrectomized rats. We conclude that, in the acutely nephrectomized living rat, 1,25-dihydroxyvitamin D3 is not present in plasma, bone, or intestine in quantities detectable by the sensitive techniques we have used. No conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 was observed during a 24-hr period after nephrectomy of vitamin D-deprived rats. This fact casts doubt upon the significance of the in vitro production of 1,25-dihydroxyvitamin D3 by nonrenal cells as an in vivo phenomenon.
Infinite flag varieties and conjugacy theorems
Authors: Peterson Dale H. , Kac Victor G.
Published: May 2006

We study the orbit of a highest-weight vector in an integrable highest-weight module of the group G associated to a Kac-Moody algebra [unk](
A
). We obtain applications to the geometric structure of the associated flag varieties and to the algebraic structure of [unk](
A
). In particular, we prove conjugacy theorems for Cartan and Borel subalgebras of [unk](
A
), so that the Cartan matrix
A
is an invariant of [unk](
A
).
Crystalline aspartate aminotransferase: lattice-induced functional asymmetry of the two subunits.
Authors: Kirsten H , Gehring H , Christen P
Published: May 2006

The enzymic activity of crystalline mitochondrial aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) was determined in suspensions of noncrosslinked microcrystals in 30% (wt/vol) polyethylene glycol. The crystals (average dimensions, 22 x 5 x 0.8 micron) were small enough to preclude diffusional rate limitation. They had the same habit as the triclinic crystals used for the determination of the spatial structure of the enzyme by x-ray crystallographic analysis [Ford, G. C., Eichele, G., and Jansonius, J. N. (1980) Proc. Natl. Acad. Sci. USA 77, 2559-2563]. Determination of the Michaelis-Menten parameters showed that the packing of the enzyme dimer into the crystal lattice not only decreases its activity but also induces a functional nonequivalence of the two subunits that behave identically in solution. The crystalline enzyme possesses a high-affinity subunit with Km values similar to those of the enzyme in solution (K'm = 0.5 mM for aspartate and 1.2 mM for 2-oxoglutarate) and a low-affinity subunit (K'm = 5.5 mM and 14.5 mM, respectively). The catalytic activity of the high-affinity subunit is 3% and that of the low-affinity subunit is 15% of the activity of the enzyme in solution. The functional asymmetry of the crystalline enzyme dimer could also be demonstrated by selective mechanism-based modification of either type of active sites. In view of the apparently identical conformation of the two subunits in the crystalline enzyme, its decreased catalytic efficiency and its functional asymmetry likely are due to constraints exerted by the crystal lattice on the conformational adaptability of the two subunits. In triclinic crystals the two subunits of the enzyme dimer have dissimilar lattice contacts.
The sequence (dC-dA)n X (dG-dT)n forms left-handed Z-DNA in negatively supercoiled plasmids.
Authors: Nordheim A , Rich A
Published: May 2006

Z-DNA-specific antibodies have been used to demonstrate the formation of left-handed Z-DNA in sequences of (dC-dA)32 X (dG-dT)32 in negatively supercoiled plasmids. Z-DNA was found to form at physiological negative superhelical densities (sigma = -0.05) in the absence of high salt concentration or chemical modifications. The binding site of the antibody to the DNA sequences was demonstrated by crosslinking experiments. (dC-dA)n X (dG-dT)n sequences are widespread in eukaryotic DNA, and possible roles for such Z elements in chromatin activation or genome rearrangements are suggested.
Covalent linking of photoreactive gonadotropin-releasing hormone to gonadotropes produces a prolonged signal.
Authors: Hazum E , Keinan D
Published: May 2006

A bioactive, photoreactive derivative of gonadotropin-releasing hormone (GnRH; gonadoliberin), [azidobenzoyl-D-Lys6]GnRH, was shown to bind covalently to dispersed pituitary cells after irradiation. Approximately 7% of the total cell-associated radioactivity was covalently bound to the receptors. Photolysis of cultured pituitary cells in the presence of the photoreactive derivative resulted in persistent activation of luteinizing hormone (LH; lutropin) release. This persistent response was time dependent and concentration dependent. No increase in the basal rate of LH release was observed with cells incubated in the presence of photoreactive GnRH analog and maintained in the dark or with hormone derivatives that lack the photoreactive azido group. These results suggest that only the covalently bound cell surface receptors account for the persistent activation of LH release after photolysis.
Vasopressin induces selective desensitization of its mitogenic response in Swiss 3T3 cells.
Authors: Collins M K , Rozengurt E
Published: May 2006

Prior incubation of quiescent cultures of Swiss 3T3 cells with vasopressin leads to loss of mitogenic stimulation on its subsequent addition in the presence of a synergistic growth factor. This desensitization is selective for vasopressin, requires prolonged incubation (half-maximal desensitization after 12 hr of treatment) for its induction, and is reversed after a 48-hr incubation in the absence of vasopressin. It is elicited by concentrations of vasopressin, and several analogues, similar to those required for stimulation of DNA synthesis. Inhibition of 125I-labeled epidermal growth factor binding and stimulation of 86Rb+ uptake by vasopressin are also selectively decreased in the refractory cells. The vasopressin receptors that mediate mitogenesis in Swiss 3T3 cells are of the pressor type, not coupled to adenylate cyclase. These cells bind [3H]vasopressin in a specific and saturable (Kd = 1 X 10(-8) M) manner. The receptors are down-regulated after prolonged vasopressin treatment; however, this cannot provide a complete explanation of desensitization because cells that are completely refractory to vasopressin retain 60% of their [3H]vasopressin binding sites. Vasopressin refractoriness must therefore occur partly at a post-receptor locus.
Novel B family sequence from an early chorion cDNA library of Bombyx mori.
Authors: Lecanidou R , Eickbush T H , Rodakis G C , Kafatos F C
Published: May 2006

A cDNA library has been constructed from transcripts present during the early stages of choriogenesis in the silk-moth, Bombyx mori. Clones representing novel sequences were selected by screening the library with (i) homotypic cDNA, (ii) rDNA and typical chorion probes of the A and B multigene families and (iii) selected clones from the library, which were positive in step (i) but negative in step (ii); this third step identified clones that hybridized to distinct subsets of the library. The sequence of one such clone was determined and shown to represent a novel subfamily of the B chorion multigene family, exclusively expressed during the early choriogenic period.
beta-Carbolines enhance shock-induced suppression of drinking in rats.
Authors: Corda M G , Blaker W D , Mendelson W B , Guidotti A , Costa E
Published: May 2006

By using Vogel's method to test the anxiolytic action of benzodiazepines and reducing the intensity of the current delivered to the drinking tube, it is possible to distinguish the pharmacological activity of three types of ligands for the benzodiazepine recognition site. An anticonflict action typical of anxiolytic benzodiazepines, a proconflict action typical of many beta-carbolines, including FG 7142 (beta-carboline-3-carboxylic acid ethyl ester methyl amide), and an antagonistic action of the proconflict and anticonflict actions typical of RO 15-1788 (ethyl-8-fluoro-5, 6-dihydro-5-methyl-6-oxo-4H-imidazol[1,5-alpha]-[1, 4]-benzodiazepine-3-carboxylate) and CGS 8216 (2-phenylpyrazolo[4,3-c]quinolin-3-(5H)-one). Pentylenetetrazole, which causes convulsions by interacting with a subunit of the gamma-aminobutyric acid receptor that is different from the benzodiazepine recognition site, also induces a proconflict action that is antagonized by anxiolytic benzodiazepines but not by RO 15-1788.
A proteolytic artifact associated with the lysis of bacteria by egg white lysozyme.
Authors: Oliver C N , Stadtman E R
Published: May 2006

Polyacrylamide gel electrophoresis of cell-free extracts of Escherichia coli that had been grown in a medium containing 32Pi disclosed the presence of several 32P-labeled proteins. Comparison of the electrophoretic patterns obtained in the presence of carrier unlabeled purified E. coli glutamine synthetase before and after treatment with trypsin, subtilisin, or snake venom phosphodiesterase showed that most of the 32P was present in the adenylyl moieties of adenylylated glutamine synthetase. Low molecular weight 32P-labeled degradation products of glutamine synthetase were also observed in extracts prepared by treatment of cells with lysozyme but not in extracts prepared by sonic oscillation. The degradation of glutamine synthetase in lysozyme-prepared extracts is likely due to an intrinsic proteolytic activity of egg white lysozyme. Proteolysis probably occurs at the esterase site of lysozyme described by Piszkiewicz and Bruice [Piszkiewicz, D. & Bruice, T.C. (1968) Biochemistry 7, 3037-3047]. Selective carboxymethylation of lysozyme histidine-15 leads to simultaneous loss of esterase and protease activities but only to partial loss of lytic activity. In view of these findings, caution is needed in the interpretation of results obtained with extracts of cells prepared by lysozyme treatment, especially when such extracts are used to investigate the properties of proteolytic enzymes.
Loss of cholesterol 7 alpha-hydroxylase activity in vitro in the presence of bivalent metal ions and by dialysis of rat liver microsomes.
Authors: Sanghvi A , Grassi E , Diven W
Published: May 2006

A loss in cholesterol 7 alpha-hydroxylase activity [cholesterol 7 alpha-monooxygenase; cholesterol,NADPH:oxygen oxidoreductase (7 alpha-hydroxylating), EC 1.14.13.17] was seen when rat liver microsomes were incubated in the presence of Ca2+, Mg2+, or Mn2+. The loss in enzyme activity was complete within only 5 min of incubation with Ca2+ and Mn2+, whereas Mg2+ required 10 to 15 min of incubation with microsomes to produce a similar inhibition. This effect of metal ions could be blocked if the incubations were carried out in phosphate buffer. Similarly, preincubation of microsomes in the presence of NaF completely prevented the loss in enzyme activity due to Ca2+ and Mg2+ ions, but only partially the loss due to Mn2+. These results suggest metal ion activation of an endogenous microsomal phosphatase, which in turn may inactivate cholesterol 7 alpha-hydroxylase through its dephosphorylation. Further, a dialyzable microsomal factor appears to be essential for stabilizing the enzyme, because dialysis of a microsomal suspension results in a considerable loss of enzyme activity.
Mutator strains of Escherichia coli, mutD and dnaQ, with defective exonucleolytic editing by DNA polymerase III holoenzyme.
Authors: Echols H , Lu C , Burgers P M
Published: May 2006

The closely linked mutD and dnaQ mutations confer a vastly increased mutation rate on Escherichia coli and thus might define a gene with a central role in the fidelity of DNA replication. To look for the biochemical function of the mutD gene product, we have measured the 3' leads to 5' exonucleolytic editing activity of polymerase III holoenzyme from mutD5 and dnaQ49 mutants. The editing activities of the mutant enzymes are defective compared to wild type, as judged by two assays: (i) decreased excision of a terminal mispaired base from a copolymer substrate and (ii) turnover of dTTP to dTMP during replication with a phage G4 DNA template. Thus, the mutD (dnaQ) gene product is likely to control the editing (proofreading) capacity of polymerase III holoenzyme.
Mapping of the genes encoding the HLA-DR alpha chain and the HLA-related antigens to a chromosome 6 deletion by using genomic blotting.
Authors: Erlich H A , Stetler D , Saiki R , Gladstone P , Pious D
Published: May 2006

We have used genomic blotting with DNA from a human cell line that has a small deletion on chromosome 6 (6.3.6) and from its parent cell line (T5-1) to map DNA fragments complementary to cloned DNA sequences encoding the HLA-B7 antigen (class I) and the alpha chain of the HLA-DR antigen (class II). The 6.3.6 variant fails to express the HLA-A, -B, -C, and -DR and MB specificities associated with one of the parental T5-1 haplotypes and has a visible deletion in the short arm of one chromosome 6 (1). The gene locus assignment was based on the expectation that, if the chromosomal location of the DNA sequences used as a hybridization probe were within the deletion, then the relative amount or size (or both) of genomic restriction fragments that hybridize to the probe in T5-1 and in 6.3.6 DNAs should differ predictably. By comparing the genomic blot patterns from T5-1 and 6.3.6 DNAs, we have shown directly that the loss of haplotype expression was due to deletion of the structural genes and have mapped the structural gene for the HLA-DR alpha chain to the chromosomal location (6p2105-6p23) defined by the 6.3.6 deletion. A cDNA clone encoding the alpha chain of the HLA-DR antigen hybridized to two genomic fragments, 4.2 and 3.8 kilobases long, generated by Bgl II digestion of T5-1 DNA. The 4.2-kilobase fragment was absent from DNA derived from the 6.3.6 deletion variant. Thus, this fragment could be assigned to the parental chromosome 6 with the A1, B8, DR3 haplotype, and the 3.8-kilobase fragment, to the chromosome 6 with the A2, B27, DR1 haplotype. In addition, comparison of the T5-1 and 6.3.6 genomic blot patterns obtained with the HLA-B7 probe revealed dosage differences for all of the class I genomic fragments generated by BamHI digestion, suggesting that all of the class I loci map to the region 6p2105-6p23.
Organization of actin cytoskeleton in normal and regenerating arterial endothelial cells.
Authors: Gabbiani G , Gabbiani F , Lombardi D , Schwartz S M
Published: May 2006

The distribution of actin stress fibers in normal and regenerating (after endothelial denudation by means of a balloon catheter) rabbit aortic endothelial cells has been studied by means of immunofluorescence with human actin autoantibodies on en face endothelial cell preparations. Our results show that: (i) under normal conditions actin is accumulated as a network at the periphery of endothelial cells. Stress fibers are present only in endothelial cells located immediately below intercostal artery branches; (ii) stress fibers develop in endothelial cells early during regeneration and persist after the end of endothelial mitotic and motile activities; and (iii) the orientation of stress fibers within the cytoplasm follows the direction of blood flow, with the exception of stress fibers situated in cells at the edge of the wound, when endothelial cell progression toward the denuded area as well as mitotic activity have ceased. We conclude that stress fibers are an organelle present in endothelial cells in vivo and that they reorganize during endothelial cell adaptation to unfavorable or pathological situations.
Arachidonic acid deficiency in streptozotocin-induced diabetes.
Authors: Holman R T , Johnson S B , Gerrard J M , Mauer S M , Kupcho-Sandberg S , Brown D M
Published: May 2006

Fatty acid compositions of phospholipids of heart, liver, kidney, aorta, and serum from rats having streptozotocin-induced diabetes were determined and compared with those of nondiabetic controls. Linoleic and dihomo-gamma-linolenic acids were increased whereas arachidonic acid was decreased in most tissues, suggesting an impairment of delta 5-desaturase activity. Acids derived from linolenic acid were increased in some diabetic tissues from diabetic animals although the linolenic content was normal, indicating less impairment in the desaturation of the omega 3 series of fatty acids. Diabetes suppressed all polyunsaturated acids in the whole animal, but the competition between omega 3 and omega 6 acids favored the excessive suppression of long-chain omega 6 acids and an increase in the proportion of omega 3 acids in lipids of vital tissues. These changes in fatty acid composition of the phospholipids may have significant effects on cellular functions and vasoregulatory control mechanisms in diabetes.
Prothrombinase complex assembly on the platelet surface is mediated through the 74,000-dalton component of factor Va.
Authors: Tracy P B , Mann K G
Published: May 2006

The blood coagulation protein factor Va forms the receptor for the serine protease factor Xa on the platelet surface. This membrane-bound complex of factor Va and factor Xa plus Ca2+ comprises the prothrombinase complex, the enzyme that catalyzes the proteolytic conversion of prothrombin to the clotting enzyme thrombin. Factor Va is a two-subunit protein composed of component D (Mr = 94,000) and component E (Mr = 74,000); subunit interaction is Ca2+ dependent. Factor Va bound to platelets consists of three peptides: component D, component E, and component D'(Mr = 90,000) which appears as the result of a platelet-associated protease cleavage of component D. The present studies were undertaken to determine which peptide(s) mediates the binding of factor Va to the platelet membrane surface and which peptide(s) serves as the binding site for factor Xa. These interactions were assessed by direct measurements of radiolabeled factor Va and factor Xa binding to platelets as well as autoradiographic visualization of the factor Va peptides associated with the platelet. Experiments were performed to determine the interaction of components D and E with platelets under reaction conditions in which components D and E were present as either the intact, functional two-subunit protein or as nonfunctional discrete peptides dissociated by the addition of Na2EDTA. The results suggest that component E mediates the binding of factor Va to the platelet and also serves as the binding site for the interaction of factor Xa with platelet-bound factor Va.
Cholecystokinin-immunoreactive neurons in rat and monkey cerebral cortex make symmetric synapses and have intimate associations with blood vessels.
Authors: Hendry S H , Jones E G , Beinfeld M C
Published: May 2006

Neurons displaying cholecystokinin-like immunoreactivity (CCK neurons) in rat and monkey cerebral cortex were examined by light and electron microscopic immunocytochemistry. CCK neurons were found to be mainly bipolar cells present in all layers and in all areas of the rat cerebral cortex. CCK neurons were also found in all regions examined in monkey cortex (pre- and post-central gyri and superior parietal lobule). The somata and the dendritic processes of CCK neurons receive relatively few synapses but both symmetric and asymmetric axosomatic and axodendritic synapses were found. The majority of axon terminals displaying CCK-like immunoreactivity formed symmetric synapses, most frequently with the somata and proximal dendrites of pyramidal and nonpyramidal neurons. The somata and processes of CCK neurons were also found to establish very close nonsynaptic associations with blood vessels and with other neurons, suggesting possible roles for the peptide in the maintenance of neuronal excitability and cerebral blood flow.
Uridine diphosphate glucose breakdown is mediated by a unique enzyme activated by fructose 2,6-bisphosphate in
Solanum tuberosum
Authors: Gibson Donna M. , Shine Ward E.
Published: May 2006

In the presence of inorganic phosphate, uridine 5′-diphosphate glucose (UDPG) is specifically hydrolyzed to glucose 1-phosphate and UDP by a unique enzyme, UDPG phosphorylase. The activity of the enzyme was maximally stimulated by fructose 2,6-bisphosphate, a regulatory metabolite recently discovered in both plants and animals, and by 2-phosphoglyceric acid. At 1 μM, fructose 2,6-bisphosphate stimulated UDPG phosphorolysis 10-fold, whereas 2-phosphoglyceric acid was required at higher concentrations (100 μM) to produce a similar effect. Fructose 2,6-bisphosphate appears to increase the affinity of the enzyme for inorganic phosphate, with a change in
K
m
from 1.6 mM to 0.3 mM. The results suggest that fructose 2,6-bisphosphate participates in the regulation of other pathways of carbohydrate metabolism in addition to playing its recognized role in glycolysis and gluconeogenesis.
Tyrosyl protein kinases in normal rat liver: identification and partial characterization.
Authors: Wong T W , Goldberg A R
Published: May 2006

Rat livers were fractionated and subcellular components were assayed for tyrosyl protein kinase activity. About 60% of the kinase activity in the cytoplasm sedimented with the microsomal fraction, whereas 40% remained in the supernatant. Purification of cytosolic and microsomal kinases by ion-exchange and gel filtration chromatography resolved a major species whose molecular mass was 75 kilodaltons (referred to as TPK 75) and a minor one whose molecular mass was greater than 160 kilodaltons. Partially purified TPK 75 phosphorylated a protein of the same molecular mass on tyrosine residues. The activity associated with TPK 75 was not stimulated by growth factors and was sensitive to thiol re-agents.
Quantitation of endogenously occupied and unoccupied binding sites for 1,25-dihydroxyvitamin D3 in rat intestine.
Authors: Massaro E R , Simpson R U , DeLuca H F
Published: May 2006

The quantitative reversible dissociation of the 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-receptor complex by the mercurial reagent mersalyl was used to develop an assay for endogenously occupied and unoccupied 1,25-(OH)2D3 binding sites. Incubation of intestinal cytosol preparations in buffer containing 50 mM Tris . HCl, 300 mM KCl, and 1.5 mM EDTA, pH 7.4, with 1 mM mersalyl for 60 min was effective in inhibiting 98% of 1,25-(OH)2D3 specific binding activity. Dissociation of bound 1,25-(OH)2[26,27-3H]D3 from the hormone-receptor complex approached completion by 180 min. In cytosol incubated with saturating levels of nonradioactive hormone, 96% of total binding activity was measurable with the hormone binding assay after displacement of bound nonradioactive ligand with 1 mM mersalyl. Endogenously occupied 1,25-(OH)2D3 binding sites contributed 0, 9, and 19% of total binding activity in rats with plasma 1,25-(OH)2D3 levels averaging 2, 121 +/- 36 and 516 +/- 92 pg/ml, respectively. Therefore, the major fraction of cytosolic 1,25-(OH)2D3 specific binding activity is unoccupied in rat intestine. The results suggest that only a small proportion of the measurable receptors are in the bound form to provide maximal 1,25-(OH)2D3-induced calcium transport.
In vitro synthesis of biologically active human leukocyte interferon in a RNA-dependent system from Saccharomyces cerevisiae
Authors: Chanda Pranab K. , Kung Hsiang-Fu
Published: May 2006

RNA isolated from yeast
Saccharomyces cerevisiae
containing recombinant plasmid DNA coding for a human leukocyte interferon gene was used as template in a modified yeast cell-free translation system. Radioactive as well as biologically active leukocyte interferon was synthesized.
Unusual human interferons produced by virus-infected amniotic membranes.
Authors: Duc-Goiran P , Robert-Galliot B , Chudzio T , Chany C
Published: May 2006

Interferon (IFN) induced in the human amniotic membrane contains at least five different molecular species, as shown by analysis in NaDodSO4/polyacrylamide gels after heating and under reducing conditions. Three of the IFN components reported here--migrating at 26, 43, and 80 kilodaltons--are of unusual antigenic structure because they are neutralized to about the same extent by anti-IFN-alpha and anti-IFN-beta antibodies. The 15- to 17-kilodalton species belongs to the IFN-alpha group, while the 21- to 22-kilodalton species, the most frequently detected major peak, is IFN-beta. In addition to their unusual size and antigenic structure, these IFNs could play a role during embryonic development and in the immune tolerance of the mother with regard to the fetus.
Biological action of epidermal growth factor and its functional receptors in normal mammary epithelial cells.
Authors: Taketani Y , Oka T
Published: May 2006

Normal murine mammary epithelial cells possessed the capacity to bind epidermal growth factor (EGF) in a specific and saturable manner. Scatchard plot analysis showed a curvilinear pattern. Assuming that this is not due to negative cooperativity of EGF binding, the data suggest the presence of two classes of receptors with a high and a low affinity: Kd values = 1 x 10(-10)M and 3.6 x 10(-9)M, respectively. The number of high- and low-affinity receptors was estimated to be 800 per cell and 8,600 per cell, respectively. The occupancy of EGF receptors for a half-maximal stimulation of DNA synthesis or inhibition of casein synthesis was about 10% and 6% of total receptors, respectively. A potent tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, acted synergistically with EGF in terms of stimulation of DNA synthesis but not in terms of inhibition of casein synthesis when the two agents were added at a suboptimal concentration. The presence of the tumor promoter increased the amount of EGF bound to mammary cells in culture and also decreased a loss in the amount of EGF in the culture medium. These results indicate that mammary epithelial cells possess functional receptors for EGF, which are modulated by the tumor promoter.