bluuebunny/crossref_metadata_2025 huggingface dataset:
Transport of D-glucose by membrane vesicles from normal and avian sarcoma virus-transformed chicken embryo fibroblasts.
Authors: Decker S , Lipmann F
Published: May 2006

Transport of D-glucose was examined in membrane vesicles from normal and avian sarcoma virus (ASV)-transformed chicken embryo fibroblasts. The initial rates of D-glucose transport were found to be 3- to 5-fold higher for vesicles from glucose-starved normal cells and ASV-transformed cells when compared with transport rates for vesicles from normal cells and serum-starved normal cells. Cytochalasin B, phloretin, and diethylstilbestrol inhibited the initial rate of transport in all types of vesicles, and 2-deoxyglucose, 3-O-methylglucose, and galactose were competitive inhibitors. At D-glucose concentrations between 0.25 and 5 mM, vesicles from normal and ASV-transformed cells displayed saturation kinetics with a Km value of 5 mM for both types of vesicles, with transformed cell vesicles showing a 3-fold increase in Vmax compared with normal cell vesicles. At D-glucose concentrations between 5 and 25 mM the initial rate of D-glucose transport was proportional to D-glucose concentration. The vesicles also showed an inhibitor-sensitive efflux at rates similar to those observed for influx.
DNA sequence of a major human leukocyte interferon gene.
Authors: Lawn R M , Gross M , Houck C M , Franke A E , Gray P V , Goeddel D V
Published: May 2006

The gene for human leukocyte interferon alpha 2 (designated either LeIF A or HuIFN-alpha 2) has been isolated from a human genome library. The DNA sequence of this gene demonstrates that it lacks introns. The 3' noncoding sequences of the IFN-alpha 2 gene correspond to two types of IFN-alpha 2 cDNA clones we have isolated that have alternate sites of polyadenylylation. A comparison of seven human IFN-alpha sequences shows that they are homologous in the 5' flanking region and contain identical "TATA box" sequences. The recombinant lambda clone containing the IFN-alpha 2 gene also contains two copies of the "Alu family" repeat sequence.
Staphylococcal alpha-toxin: oligomerization of hydrophilic monomers to form amphiphilic hexamers induced through contact with deoxycholate detergent micelles.
Authors: Bhakdi S , Füssle R , Tranum-Jensen J
Published: May 2006

Native staphylococcus aureus alpha-toxin is secreted as a hydrophilic polypeptide chain of Mr 34,000. The presence of deoxycholate above the critical micellar concentration induced the toxin monomers to self-associate, forming ring or cylindrical oligomers. The oligomers were amphiphilic and bound detergent. In deoxycholate solution, the protein-detergent complexes exhibited a sedimentation coefficient of 10.4 S. A Mr of 238,700 was determined by ultracentrifugation analyses at sedimentation equilibrium. Because quantitative detergent-binding studies indicated a protein/detergent ratio of approximately 5:1 (wt/wt), the protein moiety in each protein-detergent complex was determined to be approximately Mr 200000, corresponding to a hexamer of the native molecule. The amphiphilic toxin hexamers were ultrastructurally indistinguishable from the cytolytic, annular toxin complexes that form on and in biological target membranes. They bound lipid and could be incorporated into artificial lecithin lipid vesicles. The transition of toxin protein molecules from a hydrophilic monomer to an amphiphilic oligomer through self-association has thus been shown to be inducible solely through contact of the native protein molecules with an appropriate amphiphilic substrate.
A second pathway of leukotriene biosynthesis in porcine leukocytes.
Authors: Maas R L , Brash A R , Oates J A
Published: May 2006

Incubation of suspensions containing polymorphonuclear and eosinophilic leukocytes with arachidonic acid led to the formation of two pairs of diastereomeric 8,(15S)-dihydroxy-5,9,11,13-icosatetraenoic acids and two erythro-14,15-dihydroxy-5,8,10,12-icosatetraenoic acids. The structures were elucidated by ultraviolet spectroscopy and gas chromatography--mass spectrometric analysis of several derivatives of each compound, catalytic hydrogenation, oxidative ozonolysis with steric analysis of alcohols, and comparison to reference compounds prepared by chemical synthesis. Experiments with 18O2 and H218O indicated that in all six compounds the hydroxyl group at C-15 was derived from molecular oxygen. Two of the diastereomeric 8,15-dihydroxy acids incorporated H218O at C-8, while the other two 8,15-dihydroxy products (also diastereomers) and the 14,15-dihydroxy compounds (geometric isomers) incorporated 18O2 at C-8 and C-14, respectively, in addition to C-15. Two of the 8,15-dihydroxy acids are formed by reaction of water with an unstable allylic epoxide intermediate, (14S,15S)-oxido-5,8,10,12-icosatetraenoic acid; the two 14,15-dihydroxy acids are proposed to be formed by reaction of activated molecular oxygen with the same epoxide, which in turn originates via 15S oxygenation of arachidonic acid.
Control of expression of histocompatibility antigens (H-2) and beta 2-microglobulin in F9 teratocarcinoma stem cells.
Authors: Croce C M , Linnenbach A , Huebner K , Parnes J R , Margulies D H , Appella E , Seidman J G
Published: May 2006

Murine teratocarcinoma stem cells, unlike most other cell types, do not express major histocompatibility antigens. The steady-state levels of beta 2-microglobulin and H-2 mRNA from F9-derived teratocarcinoma stem and differentiated cells were examined by blot hybridization using cloned DNA probes specific for these mRNAs. No H-2- or beta 2-microglobulin-specific RNA was detected in F9 teratocarcinoma stem cells (clone 12-1); thus, F9 teratocarcinoma stem cells (clone 12-1) contain no more than 1/10 the H-2 and beta 2-microglobulin mRNAs of the differentiated daughter cells (clone 12-1a). We suggest that this regulation of major histocompatibility antigen expression is due to transcriptional control of the major histocompatibility antigen genes, H-2 and beta 2-microglobulin. The transcriptional regulation of these genes is accompanied by a change in their DNase I sensitivity. Normally, transcriptionally inactive genes are DNase I resistant, while active genes are DNase I sensitive. In contrast, the silent major histocompatibility antigen genes of teratocarcinoma stem cells are more DNase I sensitive than the active genes of the differentiated cells.
Distinctive urinary odors governed by the major histocompatibility locus of the mouse.
Authors: Yamaguchi M , Yamazaki K , Beauchamp G K , Bard J , Thomas L , Boyse E A
Published: May 2006

It has been shown that major histocompatibility complex (MHC) types affect the mating choices of mice and that mice can be trained to distinguish arms of a Y maze scented by odors from MHC-congeneic mice. It is now shown that sensory discrimination of MHC types by trained mice in the Y maze is equally effective with urine as the source of odors. Trained mice, male and female, successfully distinguished between urines of MHC-dissimilar F2 segregants of an MHC-congeneic cross but not between urines of MHC-identical F2 segregants. In a control study with a transfer of training procedure, in which reward was withheld to eliminate any basis for new learning, the trained mice successfully distinguished between urines from panels of MHC-congeneic inbred and F2 segregant congeneic mice that had not previously been used as urine donors. Thus urine, which is a source of chemosensory signals in many species, is also a potent source of the MHC-determined odors that distinguish individual mice.
Properties of the gastric proton pump in unstimulated permeable gastric glands.
Authors: Malinowska D H , Koelz H R , Hersey S J , Sachs G
Published: May 2006

It was found that digitonin-permeabilized resting gastric glands retained considerable acid-secretory ability. Oligomycin abolished this, and ATP was able to bypass this inhibition and restore acid secretion. Moreover, the effect of anoxia was also bypassed by ATP in these preparations. As in intact glands, acid secretion was K+ dependent, and the concentration for half-maximal effect was 18.5 +/- 1.76 mM in Na+-free solutions, a value similar to that found for resting intact glands. The slight inhibition of ATP-dependent secretion by either valinomycin or 2,4-dinitrophenol, but total inhibition by a combination of the ionophores, is interpreted to mean that, in resting gastric glands, the in situ proton pump is electroneutral and the KCl pathway supplying K+ to the luminal face of the pump is probably electroneutral.
Protein dynamics by solid-state NMR: aromatic rings of the coat protein in fd bacteriophage.
Authors: Gall C M , Cross T A , DiVerdi J A , Opella S J
Published: May 2006

The motions of the aromatic amino acids of the fd bacteriophage coat protein are described by solid-state 2H, 13C, and 15N NMR. Tryptophan-26 is immobile on time scales as slow as 10(3) HZ. The phenylalanine and tyrosine rings undergo 180 degree flips about the C beta--C gamma bond axis more often than 10(6) HZ as well as small-amplitude rapid motions in other directions.
Characterization of pro-opiocortin-converting activity in purified secretory granules from rat pituitary neurointermediate lobe.
Authors: Loh Y P , Gainer H
Published: May 2006

Lysates of secretory granules from rat pituitary neurointermediate lobes were incubated with [3H]arginine- or [3H]phenylalanine-labeled toad pro-opiocortin. The processed products formed were identified by immunoprecipitation with adrenocorticotropin (ACTH) and endorphin antisera and by migration behavior on acid/urea/polyacrylamide gels. Pro-opiocortin was cleaved by the proteolytic activity in the secretory granule fraction to approximately 21,000 Mr ACTH, approximately 13,000 Mr ACTH, alpha-melanotropin, 16,000 Mr NH2-terminal glycopeptide, beta-lipotropin, and an endorphin-related peptide. Characterization of this pro-opiocortin-converting activity shows that it (i) is present in membrane and soluble fractions of the granule lysates, (ii) has a pH optimum of 5.0, (iii) appears to cleave at pairs of basic amino acid residues in the precursor, and (iv) is inhibited by leupeptin, pepstatin A, and p-chloromercuribenzoate but not diisopropyl fluorophosphate, N alpha-p-tosyl-L-lysine chloromethyl ketone hydrochloride, chloroquine, or EDTA. These inhibitor studies suggest that the converting-enzyme activity is due to an acid thiol, arginyl protease, distinct from any known cathepsin B-like activity.
A monoclonal antibody specific for mouse dendritic cells.
Authors: Nussenzweig M C , Steinman R M , Witmer M D , Gutchinov B
Published: May 2006

Dendritic cells (DC) are a small subpopulation of lymphoid cells with distinctive cytologic features, surface properties, and functions. This report describes the DC-specific antibody (Ab) secreted by clone 33DI. Rat spleen cells immune to mouse DC were fused to the P3U myeloma. Hybrid culture supernatants were screened simultaneously against DC, a macrophage (M phi) cell line, and mitogen-stimulated lymphoblasts. 33DI Ab specifically killed 80-90% of DC from spleen and lymph node, but no other leukocytes, including Ia+ and Ia- M phi (Ia, I-region-associated antigen,). Quantitative binding studies with 5H-labeled 33D1 Ab showed that DC had an average of 14,000 binding sites per cell. Binding to DC was inhibited with Fab fragment of 33D1 Ab but not with a panel of other monoclonal antibodies, including anti-Ia Ab. Adherence and flotation procedures that enriched for DC enriched for 3H-labeled 33D1 Ab binding in parallel. 33D1 antigen was not detectable on: M phi from spleen, peritoneal cavity, and blood; three M phi cell lines; lymphocytes; granulocytes; platelets; and erythroid cells. DC continued to express the 33D1 antigen after 4 days in culture, whereas M phi and lymphocytes did not acquire it. Quantitative and autoradiographic studies confirmed that spleen and lymph node suspensions contain less than 1% DC. We conclude that 33D1 Ab detects a stable and specific DC antigen and can be used to monitor DC content in complex lymphoid mixtures.
Apolipoprotein B (B-48) of rat chylomicrons is not a precursor of the apolipoprotein of low density lipoproteins.
Authors: Van't Hooft F M , Hardman D A , Kane J P , Havel R J
Published: May 2006

We have found that, in chylomicrons from intestinal lymph fistulas in the rat, the sole molecular species of apolipoprotein B (apo B) is B-48. This protein is analogous to the B-48 apoprotein of human chylomicrons. In contrast, preparations of chylomicrons from blood serum are known to contain species of B apolipoproteins of higher molecular weight, presumably due to the presence of hepatogenous lipoproteins. We studied the removal of 125I-labeled apo B-48 of intestinal lymph chylomicrons from blood plasma of rats. The removal of [14C]cholesteryl esters of biologically labeled chylomicrons was unaffected by radioiodination. The labeled cholesteryl esters and apo B-48 disappeared rapidly from the density (P) less than 1.006 g/ml fraction of plasma. In contrast to the apo B of very low density lipoproteins (1.019 less then p less then 1.063 g/ml) after 1 hr. No 125I was found in the B-100 or B-95 apolipoproteins at any time. We conclude that, unlike the species of apo B found uniquely in hepatogenous very low density lipoproteins, the apo B-48 protein of chylomicrons is not a precursor of the B apoprotein of low density lipoproteins.
Clonal inheritance of the pattern of DNA methylation in mouse cells.
Authors: Stein R , Gruenbaum Y , Pollack Y , Razin A , Cedar H
Published: May 2006

DNA-mediated gene transfer was used to investigate the mode of inheritance of 5-methylcytosine in mouse L cells. Unmethylated phi X174 replicative form DNA remains unmethylated after its introduction and integration into these cells. On the other hand, phi X174 replicative form DNA that was methylated in vitro at its C-C-G-G residues retains these methylations as shown by restriction enzyme analysis with Hpa II and Msp I to detect methylation at this specific site. Although these unselected methylated vectors are prone to lose 30-40% of their methyl moieties upon transfection, this demethylation appears to be random. Once established, the resulting methylation pattern is stable for at least 100 cell generations. In order to examine the specificity of methylation inheritance, fully hemimethylated duplex phi X174 DNA was synthesized in vitro from primed single-strand phi X174 DNA by using 5-methyl deoxycytidine 5'-triphosphate. This molecule was inserted into mouse L cells by cotransformation and subsequently was analyzed by a series of restriction enzymes. Only methylations located at C-G residues were conserved after many generations of cell growth. The results suggest that the inheritance of the cellular DNA methylation pattern is based on a C-G-specific methylase that operates on newly replicated hemimethylated DNA.
Heat-stable inhibitor of translation in reticulocyte lysates.
Authors: de Haro C , Manne V , de Herreros A G , Ochoa S
Published: May 2006

Inhibition of translation in hemin-containing reticulocyte lysates by catalytic subunit (cS) preparations of cAMP-dependent protein kinase from bovine heart, reported earlier by our group, is due to a highly active heat-stable protein contaminant (HS). The specific activity for translational inhibition goes up by a factor of 10 when cS is heated for 10 min at 80 degrees C, which completely destroys histone phosphorylation activity. HS has been purified to homogeneity from bovine heart. It consists of a single polypeptide chain (Mr approximately 68,000). HS inhibits translation with biphasic kinetics similar to those of hemin deficiency and induces pronounced phosphorylation of the alpha subunit of the eukaryotic initiation factor eIF-2. The inhibition is relieved by eIF-2 or GTP but not by high concentrations of double-stranded RNA, thus ruling out involvement of the double-stranded RNA-activated inhibitor. Judged by poly(U) translation, HS has no effect on chain elongation. When added to crude preparations of the proinhibitor form (proHCI) of the heme-controlled translational inhibitor (HCI), HS appears to produce an increase of the HCI-to proHCI ratio. The mode of action of HS is as yet unknown.
Stereospecific chemoattraction of lymphoblastic cells by gradients of lysophosphatidylcholine.
Authors: Hoffman R D , Kligerman M , Sundt T M , Anderson N D , Shin H S
Published: May 2006

Human plasma contains chemoattractant activity for cultured cells from the mouse thymic lymphoma 6C3HED and also for lymphoblasts from concanavalin A-stimulated mouse spleen cells. A major portion of the attractant activity for both cell types could be attributed to plasma lysophosphatidylcholine. Studies on synthetic lysophosphatides showed that polar head group structure, acyl chain length, and stereochemical configuration are important determinants for attractant activity.
Impaired receptor-mediated catabolism of low density lipoprotein in the WHHL rabbit, an animal model of familial hypercholesterolemia
Authors: Bilheimer David W. , Watanabe Yoshio , Kita Toru
Published: May 2006

The homozygous WHHL (Watanabe heritable hyperlipidemic) rabbit displays either no or only minimal low density lipoprotein (LDL) receptor activity on cultured fibroblasts and liver membranes and has therefore been proposed as an animal model for human familial hypercholesterolemia. To assess the impact of this mutation on LDL metabolism
in vivo
, we performed lipoprotein turnover studies in normal and WHHL rabbits using both native rabbit LDL and chemically modified LDL (i.e., methyl-LDL) that does not bind to LDL receptors. The total fractional catabolic rate (FCR) for LDL in the normal rabbit was 3.5-fold greater than in the WHHL rabbit. Sixty-seven percent of the total FCR for LDL in the normal rabbit was due to LDL receptor-mediated clearance and 33% was attributable to receptor-independent processes; in the WHHL rabbit, essentially all of the LDL was catabolized via receptor-independent processes. Despite a 17.5-fold elevated plasma pool size of LDL apoprotein (apo-LDL) in WHHL as compared to normal rabbits, the receptor-independent FCR—as judged by the turnover of methyl-LDL—was similar in the two strains. Thus, the receptor-independent catabolic processes are not influenced by the mutation affecting the LDL receptor. The WHHL rabbits also exhibited a 5.6-fold increase in the absolute rate of apo-LDL synthesis and catabolism. In absolute terms, the WHHL rabbit cleared 19-fold more apo-LDL via receptor-independent processes than did the normal rabbit and cleared virtually none by the receptor-dependent pathway. These results indicate that the homozygous WHHL rabbit shares a number of metabolic features in common with human familial hypercholesterolemia and should serve as a useful model for the study of altered lipoprotein metabolism associated with receptor abnormalities. We also noted that the
in vivo
metabolic behavior of human and rabbit LDL in the normal rabbit differed such that the mean total FCR for human LDL was only 64% of the mean total FCR for rabbit LDL, whereas human and rabbit methyl-LDL were cleared at identical rates. Thus, if human LDL and methyl-LDL had been used in these studies, the magnitude of both the total and receptor-dependent FCR would have been underestimated.
Functional exchange of components between light-activated photoreceptor phosphodiesterase and hormone-activated adenylate cyclase systems.
Authors: Bitensky M W , Wheeler M A , Rasenick M M , Yamazaki A , Stein P J , Halliday K R , Wheeler G L
Published: May 2006

Previous studies have noted profound similarities between the regulation of light-activated 3',5'-cyclic nucleotide phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) in retinal rods and hormone-activated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in a variety of tissues. We report here the functional exchange of components isolated from the photoreceptor system, which displayed predicted functional characteristics when incubated with recipient adenylate cyclase systems from rat cerebral cortical and hypothalamic synaptic membranes and frog erythrocyte ghosts. We demonstrate functional exchange of photoreceptor components at each of three loci: the hormone receptor, the GTP-binding protein (GBP), and the catalytic moiety of adenylate cyclase. Illuminated (but not unilluminated) rhodopsin was fund to mimic the hormone-receptor complex, causing GTP-dependent activation of adenylate cyclase. The photoreceptor GBP complexed with guanosine 5'-[beta, gamma)imidotriphosphate (p[NH]ppG) produced a marked activation of recipient adenylate cyclase systems. Much smaller activation was observed when GBP was not complexed with p[NH]ppG. A heat-stable photoreceptor phosphodiesterase inhibitor reduced both basal and Mn2+-activated adenylate cyclase activities and this inhibition was reversed by photoreceptor GBP.p[NH]ppG. These data demonstrate a remarkable functional compatibility between subunits of both systems and furthermore imply that specialized peptide domains responsible for protein-protein interactions are highly conserved.
Possible role of ceramide in defining structure and function of membrane glycolipids.
Authors: Kannagi R , Nudelman E , Hakomori S
Published: May 2006

A possible role of ceramide in defining the carbohydrate structure of glycolipids and the expression of glycolipid function has been proposed, on the basis of our finding that the ceramide composition of "lacto-series" glycosphingolipid isolated from human erythrocytes shows a remarkable correlation with the terminal carbohydrate structure: (i) The ceramides of three glycosphingolipids with Lex (or x) determinant [Gal beta 1 leads to 4(Fuc alpha 1 leads to 3) GlcNAc] had almost exclusively 16:0 fatty acid; in contrast, the ceramide of its positional isomer H determinant had mainly 20--24:0 fatty acids. (ii) The ceramide of two glycosphingolipids with NeuAc alpha 2 leads to 6GAL structure was predominantly of 16:0 fatty acid, in contrast to that of its positional isomer NeuAc alpha 2 leads to 3Gal residue, in which the ceramide had 20--24:0 fatty acids. These results, together with our previous observation that ceramide composition of mouse lymphoma and myelocytic leukemia MI cells affects their antigenicity, suggest that ceramide structure may define the organization of glycosyltransferase for synthesis of the carbohydrate determinants and may affect the organization and orientation of the carbohydrate chain in membranes, eliciting or suppressing the reactivity to its ligand. Because these glycolipids with Lex and NeuAc alpha 2 leads to 6Gal structures are developmentally regulated and are highly expressed in certain tumors, ceramide composition may affect development, differentiation, and oncogenesis.
Subcellular localization of myosin light chain kinase in skeletal, cardiac, and smooth muscles.
Authors: Cavadore J C , Molla A , Harricane M C , Gabrion J , Benyamin Y , Demaille J G
Published: May 2006

Antibodies were elicited against turkey gizzard myosin light chain kinase (MLCK), purified by affinity chromatography on the enzyme bound to Sepharose, and used to localize myosin kinase--in rabbit fast skeletal, slow skeletal, cardiac, and smooth muscles--by indirect immunofluorescence. When studied on nitrocellulose replicas of NaDodSO4/polyacrylamide gel electrophoretograms, antibodies were specific for the Mr 140,000 MLCK of gizzard smooth muscle. By using the same technique, they were shown to recognize the Mr 140,000 MLCK and a Mr 75,000 polypeptide--presumably derived from the former by proteolysis--in rat arterial and stomach smooth muscle as well as in rat thyroid cells. The same antibodies reacted only with a Mr approximately equal to 75,000 protein from rat cardiac and skeletal muscle. Antibodies inhibited the activity of smooth and skeletal myosin kinases in an in vitro assay with approximately equal to 11 mole of antibody needed for 50% inhibition of 1 mole of gizzard enzyme. The antibodies stain vascular and gizzard smooth muscle cells with no apparent segregation of the enzyme in a specific part of the cell. In contrast, sarcomeric muscles exhibit a striated staining pattern, superimposable to the staining by antiactin antibodies. This shows that (i) antibodies are not species- or tissue-specific, (ii) they recognize kinases that differ in their molecular weight and ability to be phosphorylated, probably at the level of their common catalytic and calmodulin-binding domains, and (iii) sarcomeric muscle kinases are at least in part bound to the contractile apparatus and their distribution is restricted to a specific part of the sarcomere. This raises the possibility that myosin phosphorylation may be controlled not only by the Ca2+ concentration but also by actin-myosin interaction.
Changes in the activity and properties of trehalase during early germination of yeast ascospores: Correlation with trehalose breakdown as studied by
in vivo
13
C NMR
Authors: Thevelein Johan M. , den Hollander Jan A. , Shulman Robert G.
Published: May 2006

The regulation of trehalose breakdown during dormancy and the induction of germination in yeast ascospores was studied both by
in vivo
high-resolution NMR spectroscopy and
in vitro
assays of trehalase activity. Natural-abundance
13
C NMR spectra taken during the induction of germination with glucose and phosphate showed a rapid breakdown of part of the trehalose content. The presence of both glucose and phosphate was important for maximal trehalose breakdown. The
13
C NMR spectra showed that the externally added glucose and the internal trehalose were metabolized mainly to glycerol and ethanol. Under these conditions of nitrogen deprivation, full germination is not possible and trehalose breakdown stopped after ≈1 hr. At this moment resynthesis of trehalose occurred while glycerol and ethanol production from the exogenous glucose continued. In complex media where full spore germination can occur, trehalose breakdown was more pronounced. Measurements of trehalase activity in spore extracts made after addition of varying amounts of glucose and phosphate to the spores revealed a sudden 10-fold increase in the activity of trehalase, within the first minutes of spore germination. The activation was transient: after reaching a maximum between 5 and 10 min, the activity declined back to low values during the next hours. The increase in trehalase activity was not inhibited by cycloheximide or by anaerobic conditions. The decline in trehalase activity that occurred after the initial activation could be correlated with the extent of trehalose breakdown as measured by
13
C NMR. In addition to the increase in trehalase activity, differences in the control properties were found between the enzymes from dormant and germinating spores. Trehalase from dormant spores was strongly inhibited by ATP at a concentration of ≈0.5 mM, which corresponds with the ATP concentration found in dormant spores. On the other hand, trehalase from germinating spores was not inhibited by ATP up to the much higher ATP concentrations that are found in germinating spores. It is suggested that the low activity and the stringent ATP feedback inhibition of trehalase from dormant spores are responsible for the very slow mobilization of the huge amount of trehalose in dormant spores. Therefore, dormancy seems to be caused primarily by extreme curtailment of the energy production within the spore at one selective and primary point. The switch towards high activity and low ATP inhibition upon induction of germination is suggested to be responsible for the breaking of dormancy and for the rapid breakdown of trehalose that occurs during the initial phase of germination.
Effect of dose fractionation on the ethylnitrosourea induction of specific-locus mutations in mouse spermatogonia.
Authors: Russell W L , Hunsicker P R , Carpenter D A , Cornett C V , Guinn G M
Published: May 2006

As measured by specific-locus mutations in mouse spermatogonia, fractionating a dose of 100 mg of ethylnitrosourea per kg of body weight into doses of 10 mg/kg injected intraperitoneally at weekly intervals greatly reduces the mutation frequency compared with that from a single dose of 100 mg/kg. Because there is independent evidence that the doses of 10 and 100 mg/kg reach the germ cells in amounts directly proportional to the injected dose, the lower mutational response with the fractionated dose is attributed to repair. The induced mutation rate expected from a single 10-mg/kg dose (on the assumption that this would be 1/10th the rate observed after 10 such doses) would be only 75% of the spontaneous mutation rate. Mouse spermatogonia apparently have an efficient repair system that is effective even against a potent mutagen.
Variability of DNA methylation patterns during serial passage of human diploid fibroblasts
Authors: Reis Robert J. Shmookler , Goldstein Samuel
Published: May 2006

We have examined DNA methylation in diploid human fibroblasts, early and late in their replicative life-span. The extent of methylation of -C-C-G-G- was measured by comparison of fragment sizes after digestion with methylation-specific restriction enzyme
Hpa
II or
Msp
I, or both. Methylation of -C-C-G-G- sites in total DNA, occurring predominantly at internal (3′) cytosines, increased from 59% to 64% of sites in one cell strain at late passage, remained constant in another, and decreased in four other strains (54% to 48%, 58.5% to 48%, 55% to 51.5%, and 52% to 44.5%). Base composition analysis confirmed a substantial loss of total DNA 5-methylcytosine (mC) in one strain. Seven clonal isolates, examined at middle to late passage, ranged from 33% to 51% methylation of 3′ cytosines in -C-C-G-G- sites. Three discrete classes of highly repetitive DNA were found which contained
Msp
I sites at intervals of 45, 110, and 175 base pairs. These repeat families consistently had 70-80% of sites methylated at 3′ cytosines, in all clones and in all strains examined both at early and at late passage. Thus, altered methylation of repetitive sequences is unlikely to account for the variable -C-C-G-G- methylation observed in total DNA. When DNA from one fibroblast strain and from eight pure clones isolated from that parental culture was digested with
Msp
I or
Hpa
II followed by
Eco
RI and probed for γ-globin gene sequences, considerable interclonal and intraclonal heterogeneity was observed for methylation at four -C-C-G-G- sites in the γ-globin coding region of DNA. Therefore, the pattern of methylation in endogenous gene regions appears to undergo random drift during replication of diploid fibroblasts.
Repair and mutagenesis of plasmid DNA modified by ultraviolet irradiation or N-acetoxy-N-2-acetylaminofluorene.
Authors: Schmid S E , Daune M P , Fuchs R P
Published: May 2006

Plasmid DNA was modified in vitro to various extents with N-acetoxy-N-2-acetylaminofluorene or UV irradiation. The modified plasmid DNAs were then used to transform Escherichia coli strains having different repair capabilities. Both survival and mutagenesis frequencies of the plasmid were measured as a function of the number of lesions per plasmid molecule. The majority of N-2-acetylaminofluorene (AAF) adducts, like thymine dimers, were repaired by the excision (uvrA+-dependent) pathway. In rec+ strains, dose-dependent mutagenesis occurred in either AAF- or UV-modified plasmid DNA. This is in contrast with results obtained in recA- strains, in which only AAF adducts gave rise to a lower, but dose-dependent, mutagenesis frequency. In these recA- strains there was no UV mutagenesis. Unlike what is observed with phages, induction of the "SOS" functions by UV irradiation of the bacteria prior to transformation did not increase the survival or the mutagenesis of the plasmid.
Demonstration of active tolerance in maintenance of established islet of Langerhans allografts.
Authors: Faustman D , Hauptfeld V , Lacy P , Davie J
Published: May 2006

Streptozotocin-induced diabetes in mice can be reversed by transplantation of islets of Langerhans from histoincompatible mice if the islets are treated with anti-Ia-serum and complement before transplantation. Here we show that anti-Ia-treated islets most likely induce tolerance in the recipient animals. Daily injections of recipient-specific anti-I-J-serum (beginning 80 or more days after transplantation) and small numbers of donor splenocytes caused the prompt rejection of the islets in half of the animals; neither anti-I-J-serum nor donor splenocytes alone were effective. It is likely that rejection of established allografts after this treatment is the result of abolition of the activity of allograft-specific suppressor cells.
Insulin requirement for contraction of cultured rat glomerular mesangial cells in response to angiotensin II: possible role for insulin in modulating glomerular hemodynamics.
Authors: Kreisberg J I
Published: May 2006

One proposed role of glomerular mesangial cells is the regulation of glomerular blood flow by contraction. Alterations in the contractile activity of mesangial cells could lead to alterations in glomerular hemodynamics and then to glomerular injury. In this study, the effects of glucose and insulin on the contractile response of cloned homogeneous cultures of rat glomerular mesangial cells to angiotensin II were examined. Cells were cultured in normal-glucose medium (D-glucose at 200 mg/dl) and normal-glucose medium with added insulin (4 microgram/ml). To mimic the diabetic state, cells were cultured in high-glucose medium (D-glucose at 550 mg/dl) and high-glucose medium with added insulin. The media contained 20% fetal calf serum. Cells were grown for at least 1 wk in medium prior to contraction experiments. All clones of mesangial cells grown in the presence of additional insulin, in either normal- or high-glucose media, underwent contraction when treated with angiotensin II (0.001-10 microM). Seventy-five percent of the cells contracted. Not one contracted cell was seen in cultures grown without insulin in the medium, even when exposed to 10 microM angiotensin II. From these data, it appears that insulin may be required for the contractile response of mesangial cells to angiotensin II. Loss of contractile activity by mesangial cells in low- or no-insulin conditions (e.g., juvenile diabetes mellitus) could lead to a marked increase in glomerular blood flow, ultimately resulting in glomerulosclerosis.
Action of food restriction in delaying the aging process.
Authors: Masoro E J , Yu B P , Bertrand H A
Published: May 2006

Food restriction has long been known to prolong life in rodents, and recent studies have shown it to have antiaging effects in regard to a variety of physiologic and pathologic processes. It has been suggested that these actions of food restriction relate to the reduction of metabolic rate per unit of body mass brought about by this dietary regimen. Data are presented in this report showing that food restriction can have a marked life-prolonging action in rats without reducing caloric intake per gram of body weight. Moreover, the food-restricted rats consumed a greater number of calories per gram of body weight during their lifetimes than did the rats fed ad lib, yet they lived longer. Thus, the data in this report do not support the concept that food restriction slows the rate of aging by decreasing the metabolic rate.
Immunochemical analysis of the glucocorticoid receptor: identification of a third domain separate from the steroid-binding and DNA-binding domains.
Authors: Carlstedt-Duke J , Okret S , Wrange O , Gustafsson J A
Published: May 2006

The glucocorticoid-receptor complex can be subdivided into three separate domains by limited proteolysis with trypsin or alpha-chymotrypsin. The following characteristics can be separated: steroid-binding activity (domain A), DNA-binding activity (domain B), and immunoactivity (domain C). We have previously reported the separation of the steroid-binding domain from the DNA-binding domain by limited proteolysis of the receptor with trypsin. In this paper, we report the detection by immunochemical analysis of a third domain of the glucocorticoid receptor, which does not bind hormone. Immunoactivity was detected by using specific antiglucocorticoid receptor antibodies raised in rabbits against purified rat liver glucocorticoid receptor and the assay used was an enzyme-linked immunosorbent assay. After digestion with alpha-chymotrypsin, the immunoactive region of the receptor (domain C) was separated from the other two domains (A and B). The immunoactive fragment was found to have a Stokes radius of 2.6 nm. Further digestion with alpha-chymotrypsin resulted in separation of the immunoactive fragment to give a fragment having a Stokes radius of 1.4 nm. The immunoactive domain could be separated from the half of the glucocorticoid receptor containing the steroid-binding and the DNA-binding domains (Stokes radius, 3.3 nm), by limited proteolysis of the receptor by alpha-chymotrypsin followed by gel filtration or chromatography on DNA-cellulose.
"Nonrandom" DNA sequence analysis in bacteriophage M13 by the dideoxy chain-termination method.
Authors: Poncz M , Solowiejczyk D , Ballantine M , Schwartz E , Surrey S
Published: May 2006

We describe a rapid "nonrandom" DNA sequence analysis procedure that facilitates the nucleotide sequence determination of large contiguous regions of DNA. The method consists of cloning a restriction endonuclease fragment of interest into bacteriophage M13 followed by construction of a series of nuclease BAL-31 deletion mutants originating from a single site in M13 that is close to the DNA insert. Determination of the size of the deletion mutant is accomplished by hybridization to a complementary single-stranded probe derived from M13 containing that total insert followed by nuclease S1 treatment. Single-stranded M13-insert DNAs of progressively smaller sizes are isolated and analyzed by using a site-specific M13 DNA primer and the dideoxy chain-termination method. In this way, analysis of the DNA sequence proceeds from one end of the total insert to the other in a nonrandom fashion due to generation of a controlled overlapping set of deletion mutants.
Activation of thin-filament-regulated muscle by calcium ion: considerations based on nearest-neighbor lattice statistics.
Authors: Shiner J S , Solaro R J
Published: May 2006

We discuss the activation of thin-filament-regulated muscles by calcium ion in terms of a qualitative model based on nearest-neighbor lattice statistics. For the most part, the model takes into account only the essential features of the phenomenon--that there must be an interaction between calcium adsorption to troponin and crossbridge reaction with actin for calcium ion to activate contraction and that the relevant stationary states are nonequilibrium ones. Even so, the model predicts the following features which are seen experimentally but have generally not been considered in previous models: (i) the relative activations of stationary-state isometric force and ATPase are not equal; (ii) in general, neither activation of force nor that of ATPase is proportional to calcium adsorption to the activating sites; and (iii) the slopes of the relations between the activations and the logarithm of the calcium ion concentration generally depend on the necessary interaction between calcium ion adsorption and crossbridge reaction with actin. Thus, these relations show cooperative effects even if these is no interaction between calcium adsorption sites.
Mobility of cytoplasmic and membrane-associated actin in living cells.
Authors: Wang Y L , Lanni F , McNeil P L , Ware B R , Taylor D L
Published: May 2006

We have combined fluorescent analogue cytochemistry with fluorescence photobleaching recovery to measure the mobility of fluorescently labeled actin and other labeled test proteins microinjected into living amoebae. Bovine serum albumin, ovalbumin, and ribonuclease A have a cytoplasmic mobility, expressed as a diffusion coefficient, that is 1/2 to 1/3 of that observed in aqueous solution; 90% of the actin has a mobility 1/2 to 1/8 of that of G-actin in aqueous solution, and approximately equal to 10% of the actin has a mobility comparable to that of F-actin in aqueous solution. Therefore, no more than 10% of the actin in the cytoplasm of amoebae can exist as static filaments. Microinjection of phalloidin decreases the diffusion coefficient of the mobile component of cytoplasmic actin, and it also increases the low-mobility fraction to 50% but has no effect on the mobility of labeled ovalbumin. By comparing the mobility of actin in different parts of amoebae and by separating cytoplasm from plasmalemma-ectoplasm, we found the low-mobility fraction of actin to be enriched in the tail, along the plasmalemma-ectoplasm, and in contracted cytoplasm.
Generation of the alloreactive T-cell repertoire: interaction of T-cell genotype and maturation environment.
Authors: Gress R E , Hodes R J
Published: May 2006

The influences of the T-cell genotype and the T-cell maturation environment on generation of the T-cell alloreactive repertoire were evaluated in cytotoxic T-lymphocyte responses to Kb mutant determinants expressed by the strains B6. C-H-2bm1 and B6-H2-bm6. By constructing bone marrow chimeras using either H-2b of H-2d mice as the source of donor cells and either H-2d or H-2b irradiated mice as recipients, it was first determined whether the T-cell major histocompatibility complex genotype alone determines the alloreactive repertoire. The results of such experiments indicated that H-2b T cells that have matured in a normal H-2b environment (C57BL/6N, C57BL/10Sn) or H-2d T cells that have matured in a chimeric H-2b environment (B10.D2 leads to C57BL/10Sn) are responsive to Kbm1 and Kbm6 determinants while H-2b T cells that have matured in an H-2d chimeric environment (C57BL/10Sn leads to B10.D2) have a diminished responsiveness to H-2bm1 and are completely unresponsive to H-2bm6. These findings showed that T-cell genotype alone does not determine the alloreactive repertoire to mutant Kbm1 and Kbm6 determinants and suggested that the T-cell maturation environment plays a critical role in this process. Further studies were carried out to determine whether the T-cell maturation environment alone determines this repertoire, such that maturation in an H-2b but not in an H-2d environment is both necessary and sufficient to generate reactivity to Kbm6. Experiments in which either H-2d responder populations neonatally made tolerant to H-2b or unlabeled target blocking of normal H-2d responders were used provided evidence that T cells specific for Kbm6 mutant determinants are present in the repertoire of H-2d T cells that have matured in an H-2d environment. These findings suggest that the alloreactive T-cell repertoire is not determined by the T-cell major histocompatibility complex genotype alone or by the T-cell maturation environment alone but rather than it is the product of unique interactions between the two.
Persistent infection of Friend erythroleukemia cells with vaccinia virus.
Authors: Pogo B G , Friend C
Published: May 2006

Cultures of murine Friend erythroleukemia (FL) cells, which are chronically infected with leukemia virus, were inoculated with vaccinia virus. The yield of vaccinia virus was determined by assaying plaque-forming units in mouse L2 cells, and the yield of leukemia virus was determined by measuring reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) activity released into the culture fluid. Although no facilitation of one virus by the other was detected, persistently infected cultures were established. Electron microscopic examination revealed the presence of vaccinia and leukemia viruses in the same cell. The permanent lines of cells persistently infected with vaccinia were designated FLvac. Their morphology, growth rate, cloning efficiency, and ability to respond to the induction of erythrodifferentiation by treatment with dimethyl sulfoxide were not appreciably altered as compared to the parental FL cells. However, the persistently infected cells showed a marked decrease in tumorigenicity when assayed in DBA/2 mice. The infectious virus produced by FLvac cells and by L2 cells were indistinguishable as judged by restriction endonuclease patterns of virion DNA, structural proteins, and the activities of two virion-associated DNases. The yield of infectious vaccinia virus from FLvac cells generally declined after about 60 serial passages. Although some cell lines no longer yield infectious virus, they are resistant to challenge with vaccinia at concentrations that are cytolytic for L2 cells. The mechanism responsible for the establishment of the persistent infection remains unclear because defective particles, interferon production, and temperature-sensitive mutants have not been detected.
Microinjection into an identified axon to study the mechanism of fast axonal transport.
Authors: Goldberg D J
Published: May 2006

Microinjection into an axon of an identified invertebrate neuron is shown to be a useful technique for analyzing the mechanisms of fast axonal transport. It permits direct assessment of the effect of agents that cannot permeate the plasma membrane on the translocation of material in the axon. The actin filament depolymerizer DNase I, when injected into the axon of the Aplysia neuron R2, caused a local block of fast transport of [3H]glycoprotein. Two agents that should interfere with the functioning of actin filaments without causing extensive depolymerization, tne N-ethylmaleimide-modified nuclease S1 fragment of myosin (injected) and dihydrocytochalasin B (applied externally). had no effect. Together these results suggest that actin plays a structural role in the axonal cytoskeleton rather than a role in transport force generation, the effect of DNase I being mediated by structural disordering of the axoplasm. Experiments were also done with inhibitors of dynein, the microtubule-associated ATPase. erythro-9-[3-(2-Hydroxynonyl)]adenine blocked transport but vanadate was ineffective.
Aluminum: a requirement for activation of the regulatory component of adenylate cyclase by fluoride.
Authors: Sternweis P C , Gilman A G
Published: May 2006

Activation of the purified guanine nucleotide-binding regulatory component (G/F) of adenylate cyclase by F- requires the presence of Mg2+ and another factor. This factor, which contaminates commercial preparations of various nucleotides and disposable glass test tubes, has been identified as Al3+. In the presence of 10 mM Mg2+ and 5 mM F-, AlCl3 causes activation of G/F with an apparent activation constant of approximately 1-5 muM. The requirement for Al3+ is highly specific; of 28 other metals tested, only Be2+ promoted activation of G/F by F-.
Kinetic model for surface-active enzymes based on the Langmuir adsorption isotherm: phospholipase C (Bacillus cereus) activity toward dimyristoyl phosphatidylcholine/detergent micelles.
Authors: Burns R A , El-Sayed M Y , Roberts M F
Published: May 2006

A simple kinetic model for the enzymatic activity of surface-active proteins against mixed micelles has been developed. This model uses the Langmuir adsorption isotherm, the classic equation for the binding of gas molecules to metal surfaces, to characterize enzyme adsorption to micelles. The number of available enzyme binding sites is equated with the number of substrate and inhibitor molecules attached to micelles; enzyme molecules are attracted to the micelle due to the affinity of the enzyme active site for the molecules in the micelle. Phospholipase C (Bacillus cereus) kinetics in a wide variety of dimyristoyl phosphatidylcholine/detergent micelles are readily explained by this model and the assumption of competitive binding of the detergent at the enzyme active site. Binding of phospholipase C to pure detergent micelles is demonstrated by gel filtration chromatography. The experimentally determined enzyme-detergent micelle binding constants are used directly in the rate equation. The Langmuir adsorption model predicts a variety of the characteristics observed for phospholipase kinetics, such as differential inhibition by various charged, uncharged, and zwitterionic detergents and surface-dilution inhibition. The essential idea of this model, that proteins can be attracted and bound to bilayers or micelles by possessing a binding site for the molecules composing the surface, may have wider application in the study of water-soluble (extrinsic) protein-membrane interactions.