bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural requirements for sulfation of asparagine-linked oligosaccharides of lutropin.
Authors: Green E D , Morishima C , Boime I , Baenziger J U
Published: May 2006

Human and bovine pituitary glycoprotein hormones (lutropin, follitropin, and thyrotropin) contain varying amounts of N-acetylgalactosamine and sulfate. The sulfate on asparagine-linked oligosaccharides of bovine lutropin (bLH) is present exclusively on GalNAc in the sequence GalNAc(beta 1-4)GlcNAc(beta 1-2)Man alpha. We have examined the structural requirements for sulfation of bLH oligosaccharides by using a reconstituted cell-free system. After cleavage from the protein, oligosaccharides containing the sequence GalNAc(beta 1-4)Glc-NAc(beta 1-2)Man alpha were sulfated by enzymes in pituitary membranes. Addition of one or two sulfates was observed, depending upon the number of GalNAc acceptor sites on the oligosaccharide. Neither GalNAc alone nor oligosaccharides devoid of GalNAc were sulfated. Membranes from placenta or liver did not sulfate oligosaccharides released from bLH, indicating that the sulfating activity is pituitary-specific. The lack of peptide dependence for sulfation, in conjunction with the oligosaccharide specificity, suggests that the sequence GalNAc(beta 1-4)GlcNAc(beta 1-2)Man alpha contains the recognition signal for the sulfotransferase(s).
Amino-terminal sequence of p36 and associated p10: identification of the site of tyrosine phosphorylation and homology with S-100.
Authors: Glenney J R , Tack B F
Published: May 2006

p36 is a major substrate of both viral and growth factor-receptor-associated tyrosine protein kinases. p36 can be isolated as a complex consisting of a subunit of Mr 36,000 (p36) and a subunit of Mr 10,000 (p10), and it represents an abundant cellular protein. We have isolated the p36-p10 complex from bovine intestinal epithelium and analyzed the amino terminus of both subunits. Sequence analysis of the first 56 amino acids of p10 demonstrates a striking sequence homology (48% identically placed residues) with the Mr 10,000 calcium-binding proteins from bovine brain, termed S-100. Intestinal p36 could be effectively labeled on a single tyrosine in vitro with immunoprecipitated pp60v-src and [gamma-32P]ATP. Mild proteolysis of p36 with chymotrypsin resulted in the cleavage into large (Mr, 33,000) and small domains (Mr, 3000), with the latter representing the phosphorylated amino terminus. Although the amino terminus is apparently blocked, sequence analysis of a secondary tryptic peptide of the Mr 3000 fragment as well as the amino-terminal sequence of the Mr 33,000 domain and overlapping peptides clearly established the site of tyrosine phosphorylation.
A unique structure at the carboxyl terminus of the largest subunit of eukaryotic RNA polymerase II.
Authors: Corden J L , Cadena D L , Ahearn J M , Dahmus M E
Published: May 2006

Purified eukaryotic nuclear RNA polymerase II consists of three subspecies that differ in the apparent molecular masses of their largest subunit, designated IIo, IIa, and IIb for polymerase species IIO, IIA, and IIB, respectively. Subunits IIo, IIa, and IIb are the products of a single gene. We present here the amino acid composition of calf thymus subunits IIa and IIb and the C-terminal amino acid sequence of subunit IIa (IIo) inferred from the nucleotide sequence of part of the mouse gene encoding this RNA polymerase subunit. The calculated amino acid composition of the peptide unique to subunit IIa indicates that subunit IIa contains a domain rich in serine, proline, threonine, and tyrosine. The sequence at the 3' end of the mouse RNA polymerase II largest subunit gene reveals that the C-terminal domain consists of 52 repeats of a seven amino acid block with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This sequence is also unusual in that it contains a high percentage of potential phosphorylation sites.
Identification of calmodulin-dependent protein kinase III and its major Mr 100,000 substrate in mammalian tissues.
Authors: Nairn A C , Bhagat B , Palfrey H C
Published: May 2006

A major substrate, Mr 100,00 (100 kDa), for a Ca2+/calmodulin (CaM)-dependent protein kinase found in many mammalian tissues has been purified from rat pancreas. The purified substrate was used to identify and partially purify a CaM-dependent protein kinase (CaM kinase III) from rat pancreas. The physical properties and substrate specificity of CaM kinase III were distinct from those of all known CaM-dependent protein kinases. Only CaM kinase III was able to phosphorylate the 100-kDa protein; synapsin I, phosphorylase b, myosin light chain, and histone were poor substrates for this enzyme. Polyclonal antibodies, raised against the purified 100-kDa protein, recognized the protein in a variety of mammalian tissues and cell lines. Immunoassay revealed that the 100-kDa protein made up 0.3-1.7% of the total cytosolic protein in these samples. Analysis of CaM kinase III revealed that the enzyme had a similar widespread tissue distribution. These results demonstrate the existence of a fifth CaM-dependent protein phosphorylation system present in high levels in animal cells.
Kinetic properties of the blue-light response of stomata
Authors: Iino Moritoshi , Ogawa Teruo , Zeiger Eduardo
Published: May 2006

The stomatal response to blue light was analyzed with gas-exchange techniques in
Commelina communis
L. leaves by using high-fluence-rate short pulses. Pulses of blue light were given under a background of high-fluence-rate red light, which maintained photosynthesis at near saturation and stomatal conductance at a steady state. A single blue light pulse of 1-100 sec induced an increase in stomatal conductance, which peaked after 15 min and then returned to the initial steady-state level within 50-60 min after the pulse. The response could be repeatedly induced in the same leaf. Red light pulses on a red background did not induce any comparable response. The stomatal response quantified by integrating the conductance increases after pulse application approached saturation with increasing pulse duration (
t
½
≈9 sec with 250 μmol·m
-2
·sec
-1
of blue light). After a saturating pulse, sensitivity to a second pulse was restored slowly. This recovery response, quantified from the conductance increases caused by the two pulses, approached saturation with a
t
½
of ≈9 min. These results were used to test a model in which a molecular component in the sensory transduction process is considered to exist in two interconvertible forms,
A
and
B
. If
B
is the physiologically active form inducing stomatal opening, then
A
is the inactive form. The
A
to
B
conversion is a light-induced reaction and the
B
to
A
conversion is a thermal reaction. Rate constants for these reactions were estimated from single- and double-pulse experiments (at a fluence rate of 250 μmol·m
-2
·sec
-1
,
k
1
= 0.075 sec
-1
; thermal rate constant
k
d
= 0.0014 sec
-1
), allowing the calculation of steady-state concentration of
B
under continuous irradiation. The calculated values accurately predicted the steady-state stomatal conductances under continuous blue light.
Evolution of apolipoprotein E: mouse sequence and evidence for an 11-nucleotide ancestral unit.
Authors: Rajavashisth T B , Kaptein J S , Reue K L , Lusis A J
Published: May 2006

Apolipoprotein E (apo E) is responsible for the binding of very low density lipoprotein and chylomicron remnants to cellular receptors thereby removing them from circulation. We have isolated and determined the sequence of a cDNA encoding 285 amino acids and the entire 3' untranslated region of 112 nucleotides of mouse apo E. The remaining coding sequence was determined by sequencing mouse liver mRNA. Comparisons with rat and human apo E sequences showed a high degree of conservation although there were regions in each species that were characterized by unique insertions and deletions. Analysis of the sequence homologies within apo E revealed that the entire sequence is made up of repetitive units. The most primitive unit appeared to be an 11-nucleotide repeat within higher order repeats of 22 or 33 nucleotides. The 11-nucleotide unit -TCGGACGAGGC- is read in all three reading frames, and when tandemly repeated, it encodes the highly conserved amino acid sequence Xaa-(Glu/Asp)-(Glu/Asp)-Xaa-Arg-Xaa-Arg-Leu-Gly-Xaa-Xaa. We postulate that apo E and those other apolipoproteins related to it have arisen by duplications and subsequent modifications of this or a closely related 11-nucleotide ancestral sequence.
Characterization of the enzymatic lesion in inherited phosphofructokinase deficiency in the dog: an animal analogue of human glycogen storage disease type VII.
Authors: Vora S , Giger U , Turchen S , Harvey J W
Published: May 2006

Mammalian phosphofructokinase (PFK; ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) exists in multimolecular forms, which result from random tetramerization of three distinct subunits, M (muscle-type), L (liver-type), and P (platelet-type), each under a separate genetic control. Human muscle and liver contain homotetramers M4 and L4, respectively, whereas erythrocytes contain a mixture of M4, M3L, M2L2, ML3, and L4 isozymes. Homozygous deficiency of the M subunit in man results in glycogen storage disease (GSD) type VII, which is characterized by exertional muscle weakness and compensated hemolysis; the residual erythrocyte PFK consists of isolated L4 isozyme. Recently, PFK deficiency associated with isolated hemolytic anemia has been identified among English springer spaniel dogs. We investigated the genetic control of the dog PFK system and the nature of the enzymatic defect in two PFK-deficient animals, using chromatographic and immunological techniques. Our studies indicate the existence of a trilocus isozyme system for the dog, as is the case with other mammals. Muscle PFK consists of M4 isozyme, whereas the predominant species of liver and platelet consists, respectively, of the L4 and P4 isozyme; erythrocyte PFK consists of a three- or four-membered set composed of M and P subunits. PFK deficiency in the dogs was found to result from a total and universal lack of the M subunit, as is the case in man. However, the probands consistently exhibited L4 isozyme in their muscle; P4, L4, and hybrids thereof in their erythrocytes; and an increase in the L-containing isozymes in their platelets, indicating a generalized anomalous presence of the L subunit. The apparent absence of muscle disease in these animals is most likely accounted for by both the well-known high oxidative potential of the canine muscle in general and the presence of liver PFK in the M-deficient muscle in particular. In contrast, presence of hemolysis despite residual P4 and hybrids of P and L in the erythrocytes may be inferred to result in severe glycolytic handicap under existing intraerythrocytic conditions.
Identification of a polymorphic variant associated with HLA-DQw3 and characterized by specific restriction sites within the DQ beta-chain gene.
Authors: Kim S J , Holbeck S L , Nisperos B , Hansen J A , Maeda H , Nepom G T
Published: May 2006

Restriction endonuclease digestion of genomic DNA from 24 lymphoblastoid cell lines homozygous for the HLA class II specificity DQw3, followed by hybridization with a DQ beta-chain cDNA probe, identified a genomic polymorphism with variable BamHI and HindIII recognition sites. This restriction fragment pattern was found for several haplotypes associated with the DQw3 specificity, including some haplotypes positive for the HLA-DR specificities DR4, DR5, DRw8, and DRw12. The variant fragment pattern corresponds precisely with the reactivity of a monoclonal antibody, A-10-83, previously shown to define a serologic split of DQw3. Serologic detection of the specific DQw3.1 genomic polymorphism indicated that the corresponding DQ beta-chain variants are expressed. This polymorphic restriction fragment pattern, then, represents a selective marker for DQ beta-chain genes that appear to define a DQ beta-chain-associated specificity, here called DQw3.1.
Enhancement of antibody response by mouse dendritic cells pulsed with tobacco mosaic virus or with rabbit antiidiotypic antibodies raised against a private rabbit idiotype.
Authors: Francotte M , Urbain J
Published: May 2006

The role of splenic lymphoid dendritic cells (DC) and macrophages (M phi) from mice in induction of immune responses in vivo has been investigated. Varying numbers of purified DC and M phi pulsed in vitro with tobacco mosaic virus (TMV) or with rabbit antiidiotypic antibodies (Ab2) directed against a private rabbit anti-TMV idiotype were injected back into syngeneic mice. In both systems, DC appeared to strongly enhance the primary and secondary responses to the virus. Optimal responses were obtained with 5 X 10(4) purified DC carrying TMV or rabbit Ab2. In contrast, M phi were less efficient by a factor of at least 100. These results show the potency of lymphoid DC as inducing cells in T-dependent antibody responses in vivo.
Phagocytic cells synthesize 19-nor-10-keto-25-hydroxyvitamin D3, a metabolite that may induce differentiation of the human monoblastic cell line U937.
Authors: Gray T K , Millington D S , Maltby D A , Williams M E , Cohen M S , Dodd R C
Published: May 2006

Phagocytic cells, including normal human blood neutrophils and monocytes, metabolize 25-hydroxyvitamin D3 in vitro to more polar metabolites. Cells of the human monoblastic cell line U937 produced three metabolites when incubated with 25-hydroxyvitamin D3. One of these metabolites, previously designated peak III, has its maximal absorbance at 310 nm. Mass spectral analysis of the trimethylsilylated derivatives of peak III revealed a spectral pattern very similar to that published for the trimethylsilylated derivatives of 19-nor-10-keto-25-hydroxyvitamin D3. The biosynthetic 19-nor-10-keto-25-hydroxyvitamin D3 was active in the induction of differentiation of U937 cells.
On the mechanisms of induction of cancer-protective enzymes: a unifying proposal.
Authors: Prochaska H J , De Long M J , Talalay P
Published: May 2006

Induction of detoxification enzymes is a major mechanism whereby a wide variety of chemical agents protect rodents against neoplastic, mutagenic, and other toxicities of carcinogens. The enzyme NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) can protect against the toxicities of quinones and is a useful marker for protective enzyme induction. Quinone reductase can be induced in murine Hepa 1c1c7 hepatoma cells and 3T3 embryo fibroblasts by compounds that are chemoprotectors in vivo, including some phenolic antioxidants, azo dyes, aromatic diamines, and aminophenols. Structurally dissimilar catechols (1,2-diphenols) and hydroquinones (1,4-diphenols) induce quinone reductase in these systems, but resorcinol (1,3-diphenol) and its substituted analogues are inactive. Furthermore, only aromatic 1,2- and 1,4-diamines and aminophenols are inducers, whereas the 1,3-diamines are completely inactive. These findings suggest that the functional capacity to form quinones or quinone-diimines, rather than the precise structure, is essential for inductive activity and that the generation of the signal for enzyme induction depends upon oxidation-reduction lability. The observations that some chemoprotective compounds (e.g., azo dyes, beta-naphthoflavone) induce both cytochromes P-450 and quinone reductase, whereas others (e.g., tert-butylhydroquinone) induce only quinone reductase, can be reconciled by the fact that inducers of the first type are metabolized by P-450 enzymes to form products that are functionally similar to compounds of the second type.
The putative transforming protein of S13 avian erythroblastosis virus is a transmembrane glycoprotein with an associated protein kinase activity.
Authors: Hayman M J , Kitchener G , Vogt P K , Beug H
Published: May 2006

S13 is an avian retrovirus that transforms both fibroblasts and erythroblasts. The gene product responsible for the oncogenic effects of S13 is the env-related glycoprotein gp155. In this report we show that gp155 is a transmembrane protein with a 55-kDa cytoplasmic domain. Pulse-chase analysis shows that gp155 was cleaved posttranslationally into two glycosylated proteins, gp85 and gp70. In addition, we show that a tyrosine protein kinase activity is associated only with the gp70 protein in microsomes and in immune complexes.
Properties of the Syrian hamster phosphomannosyl receptor: an aggregate of low molecular weight proteins.
Authors: Maler T , Rosenblum B B , Jourdian G W
Published: May 2006

Phosphomannosyl receptor (PMR) isolated from Syrian hamster liver was purified to apparent homogeneity by affinity chromatography and one-dimensional PAGE. On one-dimensional PAGE, the receptor migrated with a Mr approximately equal to 215,000 as detected by a silver-staining reagent or by immunoblotting [utilizing antiPMR generated against purified hamster PMR (Mr 215,000) sequentially purified by affinity chromatography and NaDodSO4/PAGE]. On two-dimensional PAGE the receptor was partially dissociated into low-molecular weight components. The protein distribution on immunoblots of two-dimensional gels of hamster liver homogenates was nearly identical to that observed for purified hamster liver PMR. When liver homogenates were subjected to one-dimensional PAGE and immunoblotted under nonreducing conditions, an intensely labeled band that migrated with an apparent Mr of 43,000-49,000 was observed; under reducing conditions a single band with a Mr of 49,000 was observed. The low molecular weight compound was present in the soluble 130,000 X g supernatant but not in the particulate fraction of liver homogenates. An immunoreactive component of similar molecular weight was also present in hamster serum and plasma. These results suggest that PMR is either an aggregate comprised of small molecular weight components or, alternatively, that a number of small molecular weight components are tightly associated with PMR.
Control of oxidative metabolism and oxygen delivery in human skeletal muscle: a steady-state analysis of the work/energy cost transfer function.
Authors: Chance B , Leigh J S , Clark B J , Maris J , Kent J , Nioka S , Smith D
Published: May 2006

The concept of transfer function for organ performance (work output vs. biochemical input) is developed for skeletal and cardiac muscle under steady-state exercise conditions. For metabolic control by the ADP concentration, the transfer function approximates a Michaelis-Menten hyperbola. Variation of the work identifies metabolic operating points on the transfer function corresponding to ADP concentrations or to a ratio of inorganic phosphate to phosphocreatine that can be determined by phosphorus nuclear magnetic resonance. This operating point is characterized by the fraction (V/Vmax) of maximal activity of oxidative metabolism in the steady state. This quantity appears to be useful in predicting the degree to which metabolic homeostasis is effective; poorly controlled metabolic states can readily be identified and are used in the diagnosis and therapy of metabolic disease in the organs of neonates and adults.
Correlation between fusion and the developmental regulation of membrane glycoproteins in L6 myoblasts.
Authors: Rosenberg J , Szabo A , Rheuark D , Kayalar C
Published: May 2006

Expression of membrane glycoproteins in L6 myoblasts during the course of myogenesis was investigated. The effects of several inhibitors of myoblast fusion and differentiation were also studied. The predominant change in plasma membrane proteins concomitant with fusion was the reduction in the expression of a major 105-kDa glycoprotein and the appearance of a 90-kDa glycoprotein. This change was blocked by bromodeoxyuridine and two metalloendoprotease inhibitors, phenanthroline and benzyloxycarbonyl-Ser-Leu-NH2, all of which have been shown to inhibit myoblast fusion. The nature of this inhibition suggests a role for an endogenous metalloendoprotease in myoblast commitment to terminal differentiation. The possible function of the developmentally regulated glycoproteins in myogenesis is also discussed.
Transcription of glnA by purified Escherichia coli components: core RNA polymerase and the products of glnF, glnG, and glnL.
Authors: Hunt T P , Magasanik B
Published: May 2006

We have shown that the purified glnF (ntrA) product of Escherichia coli binds to core RNA polymerase. Together these proteins initiated transcription at the nitrogen-regulated promoter glnAp2 on a supercoiled template. The initiation of transcription at glnAp2 on a linear template required in addition NRI, the product of glnG (ntrC), and NRII2302, the product of a mutant allele of glnL (ntrB). These results identify the glnF product as a new sigma factor specifically required for the transcription of nitrogen-regulated and of nitrogen-fixation promoters. We propose rpoN as the proper designation for glnF, and sigma 60 for its product. Our results indicate that sigma 60 RNA polymerase recognizes the nitrogen-regulated/nitrogen-fixation promoter consensus sequence C-T-G-G-Y-A-Y-R-N4-T-T-G-C-A. Initiation of transcription in the intact cell appears to require in addition the active form of NRI, the product of glnG. Conversion of NRI to its active form is apparently brought about by NRII, the product of glnL, in response to nitrogen deprivation.
Detection of simple polygenic segregations in a natural population
Authors: Thompson James N. , Mascie-Taylor C. G. N.
Published: May 2006

Penetrance frequencies were used to quantify segregating polygenic effects in a natural population of
Drosophila melanogaster
. When males from a series of 100 isofemale strains were crossed to females from a veinlet (
ve
) line that had been selected for shortened veins, gaps commonly appeared in the fifth longitudinal (L5) vein in the
ve
/+ heterozygotes. We were able to assign each strain to one of six significantly different clusters, based upon the pattern of polygenic modifiers of
ve
dominance segregating in each strain. We conclude that the extensive range of phenotypic variation in vein-forming ability is actually based upon a relatively simple underlying polygenic structure that is consistent with segregation of only a small number of alleles or allele combinations in the wild population.
Organization and sequences of the diversity, joining, and constant region genes of the human T-cell receptor beta chain.
Authors: Toyonaga B , Yoshikai Y , Vadasz V , Chin B , Mak T W
Published: May 2006

The organization and sequences of the human beta-chain T-cell receptor diversity, joining, and constant region segments are described. The beta chain of the human T-cell receptor, analogous to the mouse counterpart, consists of two distinct constant region genes approximately equal to 10 kilobases apart. The two constant region genes, C beta 1 and C beta 2, are very similar not only in sequence but also in genomic organization. The coding sequences of each of these C beta constant region genes are divided into four exons. The first two exons encode most of the extracellular constant domain. The third exon encodes a major part of the presumed transmembrane portion, and the last exon contains the cytoplasmic coding sequence as well as 3' untranslated sequences. Except for a stretch of approximately equal to 95 highly conserved nucleotides extending 3' of the first exon of the C region genes, little homology can be found between the intron sequences of C beta 1 and C beta 2. A small cluster of joining region (J beta) gene segments is located approximately equal to 5 kilobases upstream of each of these two constant regions. The first cluster, J beta 1, contains six functional J gene segments while the second, J beta 2, contains seven functional J gene segments. In addition, diversity region (D beta) gene segments are located approximately equal to 600 base pairs upstream of each J beta. Recombinational signals containing highly conserved heptamer and nonamer sequences separated by 12 or 23 bases are found adjacent to all of these D beta and J beta gene segments. These signal sequences are thought to be involved in the somatic recombination processes. These results indicate that what appears to be a gene duplication event giving rise to these two distinct regions must have arisen a long time ago in the evolution of this gene locus.
Isolation and characterization of anti-monosialoganglioside monoclonal antibody 19-9 class-switch variants.
Authors: Steplewski Z , Spira G , Blaszczyk M , Lubeck M D , Radbruch A , Illges H , Herlyn D , Rajewsky K , Scharff M
Published: May 2006

Three different monoclonal antibody isotypes, IgG1, IgG2b, and IgG2a, were derived by selection of isotype-switch variants from the CO-19-9 hybridoma. All three antibodies retained their binding specificities and affinities and bound to the same epitope--as defined by the anti-idiotype analysis. Availability of gamma 1, gamma 2b, and gamma 2a Ig heavy chain variants directed against the same epitope on the monosialoganglioside antigen permitted detailed analysis of their Fc fragment receptor (FcR) binding affinities, their cytolytic activities (antibody-dependent, macrophage-mediated cytotoxicity) in vitro, and their tumoricidal activities in vivo. Analysis of the binding of three isotypes to the human FcR expressed by U937 cells induced by gamma interferon has shown that only IgG2a proteins bound to high-affinity FcR, but not IgG1 or IgG2b variants. Although all three isotypes were active in the antibody-dependent, macrophage-mediated cytotoxicity assay with murine thioglycolate-elicited macrophages, the IgG2a gave the highest percentage of lysis; similar results were obtained in the same assay with human monocytes as effector cells. In the nude mice experiment, only the IgG2a variant inhibited growth of human colorectal carcinoma, while IgG1 and IgG2b were ineffective. Thus, selection of isotype-switch variants resulted in the conversion of monoclonal antibody from noncytolytic to cytolytic with possible immunotherapeutic application.
Independent segregation of two antigenic specificities (VP3 and VP7) involved in neutralization of rotavirus infectivity.
Authors: Hoshino Y , Sereno M M , Midthun K , Flores J , Kapikian A Z , Chanock R M
Published: May 2006

Antiserum prepared against the M37 strain of rotavirus, recovered from an asymptomatic newborn infant in Venezuela, neutralized two prototype human rotaviruses that define two separate serotypes: serotype 1 (Wa) and serotype 4 (ST3). Thus, the M37 strain is a naturally occurring intertypic rotavirus. Analysis of reassortant viruses produced during coinfection in vitro indicated that the observed dual serotype specificity of M37 resulted from sharing a related outer capsid protein, VP3, with the ST3 virus and another related outer capsid protein, VP7, with the Wa virus. Analysis of single (VP3)-gene-substitution reassortants indicated that VP3 was as potent an immunogen as VP7. In addition, direct evidence was obtained that the serotype specificity of neutralizing antibody elicited by VP3 can differ from the serotype specificity of neutralizing antibody elicited by VP7, indicating the need for a dual system of rotavirus classification in which the neutralization specificity of both VP3 and VP7 outer capsid proteins are identified.
Two molecular weight forms of muscarinic acetylcholine receptors in the avian central nervous system: switch in predominant form during differentiation of synapses.
Authors: Large T H , Rauh J J , De Mello F G , Klein W L
Published: May 2006

Muscarinic acetylcholine receptors from the avian central nervous system were examined for developmental changes that correlated with the differentiation of cholinergic synapses. In contrast to previous studies that showed a single molecular weight form of muscarinic receptors in the mature central nervous system, the current study of receptors from embryonic and newly hatched chick retina showed the presence of two electrophoretic forms having apparent molecular weights of 86,200 +/- 400 and 72,200 +/- 300. Two receptor forms also were observed in embryonic cerebrum, optic tectum, and cerebellum. Each form was present, although decreased in molecular weight by 6000, after treatment with deglycosylating enzymes, consistent with molecular differences occurring in the protein portions, rather than the carbohydrate portions, of the molecules. The relative proportions of the high and low molecular weight receptors in retina showed a striking inversion during development. Before synaptogenesis, receptors were mainly of Mr 86,000, whereas after synaptogenesis, receptors were mainly of Mr 72,000. Development of a predominantly low molecular weight receptor population also occurred in aggregate, but not monolayer, cell culture, suggesting a possible role for cell-cell interactions in triggering the change. Pulse-chase labeling of receptors on cultured cells indicated that both forms were present on the cell surface; the labeled Mr 86,000 population had a half-life of 5 hr, whereas the labeled Mr 72,000 population had a half-life of 19 hr. The change in size of muscarinic receptors during development may reflect the action of regulatory mechanisms critical to the proper assembly and function of synapses in the central nervous system.
Purification and partial characterization of a cholinergic neuronal differentiation factor.
Authors: Fukada K
Published: May 2006

The choice of transmitter made by postmitotic rat sympathetic neurons in cell culture can be controlled by the environment in which they develop. One of the differentiation signals is a protein secreted by heart cells that can induce previously noradrenergic neurons to synthesize acetylcholine and form cholinergic synapses. This change in phenotype occurs without alteration in neuronal survival or growth. The differentiation factor has now been purified at least 100,000-fold, and it is homogeneous by several criteria. (i) The cholinergic activity comigrates with a single 125I-labeled protein band of 45 kDa in one-dimensional NaDodSO4/PAGE. (ii) The biological activity comigrates precisely with a series of five 125I-labeled protein spots of 45 kDa in two-dimensional gel electrophoresis. (iii) Treatment of the 45-kDa band with endo-beta-N-acetylglucosaminidase F reduces the apparent molecular size of both the labeled protein and the biological activity to a band of 22 kDa. The data suggest that the differentiation factor is a slightly basic glycoprotein with at least six glycosylation sites.
A synthetic peptide substrate specific for casein kinase II.
Authors: Kuenzel E A , Krebs E G
Published: May 2006

A synthetic peptide having the sequence Arg-Arg-Arg-Glu-Glu-Thr-Glu-Glu-Glu was found to serve as a convenient substrate for the protein kinase generally referred to as casein kinase II. The enzyme exhibited an apparent Km of 500 microM for the peptide, as compared to an apparent Km of 50 microM for casein. The maximum velocities for phosphorylation of the peptide and of casein were similar. The peptide was not phosphorylated by any of eight other protein kinases, all of which were shown to be active toward their known substrates. The peptide was used to monitor activity during steps in the purification of casein kinase II from bovine liver. These experiments demonstrated that with this peptide it is now possible to obtain specific measurements of casein kinase II activity in crude enzyme preparations.
3-Hydroxy-3-methylglutaryl-coenzyme A reductase is present in peroxisomes in normal rat liver cells.
Authors: Keller G A , Barton M C , Shapiro D J , Singer S J
Published: May 2006

The location inside rat liver parenchymal cells of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase; EC 1.1.1.34), the key regulatory enzyme in cholesterol biosynthesis, has been examined by immunoelectron microscopy and by subcellular fractionation. Although HMG-CoA reductase is generally thought to be exclusively a microsomal enzyme, we find that a substantial portion of cellular HMG-CoA reductase is localized in peroxisomes. Immunoelectron microscopic labeling of ultrathin frozen sections of normal rat liver, using two monoclonal antibodies to purified HMG-CoA reductase, showed that the enzyme is present in the peroxisomes at a higher concentration than at any other site inside the hepatocytes. Subcellular fractionation studies using Percoll and metrizamide gradients demonstrated a close correspondence of peaks of HMG-CoA reductase activity and of catalase activity, again revealing the presence of the reductase enzyme in peroxisomes. HMG-CoA reductase is therefore localized in peroxisomes in addition to being in the microsomal fraction.
Major deletions in the gene encoding the p53 tumor antigen cause lack of p53 expression in HL-60 cells.
Authors: Wolf D , Rotter V
Published: May 2006

The tumor antigen p53 is overproduced in transformed cells of various species, including man. HL-60 is an exceptional human tumor cell line that does not express this protein. Hybridization of polyadenylylated mRNA of these cells with a human p53 cDNA probe (p53-H14), which we cloned, had indicated a total absence of the mature-size (3.0 kilobases) or any aberrant p53 mRNA species. Analysis of the genomic HL-60 DNA indicated that the p53 gene in these cells was significantly altered. Most of the gene was deleted, and the residual p53 sequences of these cells, which show weak homology, mapped to the corresponding 5' region of the p53 gene. In agreement with previously documented results, we found that HL-60 cells have an amplified c-myc gene. We suggest that the deficiency of the p53 protein in HL-60 cells could have been overcome by using an alternative metabolic pathway. The c-myc product is a candidate for such an alternative protein.
Evolution of green plants as deduced from 5S rRNA sequences
Authors: Hori Hiroshi , Lim Byung-Lak , Osawa Syozo
Published: May 2006

We have constructed a phylogenic tree for green plants by comparing 5S rRNA sequences. The tree suggests that the emergence of most of the uni- and multicellular green algae such as
Chlamydomonas, Spirogyra, Ulva
, and
Chlorella
occurred in the early stage of green plant evolution. The branching point of
Nitella
is a little earlier than that of land plants and much later than that of the above green algae, supporting the view that
Nitella
-like green algae may be the direct precursor to land plants. The Bryophyta and the Pteridophyta separated from each other after emergence of the Spermatophyta. The result is consistent with the view that the Bryophyta evolved from ferns by degeneration. In the Pteridophyta,
Psilotum
(whisk fern) separated first, and a little later
Lycopodium
(club moss) separated from the ancestor common to
Equisetum
(horsetail) and
Dryopteris
(fern). This order is in accordance with the classical view. During the Spermatophyta evolution, the gymnosperms (
Cycas, Ginkgo
, and
Metasequoia
have been studied here) and the angiosperms (flowering plants) separated, and this was followed by the separation of
Metasequoia
and
Cycas
(cycad)/
Ginkgo
(maidenhair tree) on one branch and various flowering plants on the other.
Nick-translation of metaphase chromosomes: in vitro labeling of nuclease-hypersensitive regions in chromosomes.
Authors: Kuo M T , Plunkett W
Published: May 2006

Chinese hamster metaphase chromosomes were labeled by nick-translation, which involved pretreatment of metaphase chromosomes with low levels of DNase I followed by incubation with DNA polymerase I and radioactively labeled nucleotides. The labeled DNA was located on nuclease-hypersensitive regions of the chromosomes, as suggested by the following observations. (i) The labeled DNA was hypersensitive to the subsequent DNase I digestion. (ii) The labeled DNA contained no nucleosomes. DNA reassociation kinetic analysis suggested that the labeled DNA was enriched in repetitive DNA sequences. Base composition analyses showed that the labeled DNA was highly enriched in guanine and adenine residues, suggesting that the nick-translation reaction was asymmetrical and the strand enriched in purine was preferentially translated. Autoradiographic analysis revealed that the label was distributed on every chromosome, but there was a lower grain density on the Y chromosome, which is heterochromatic and exhibits a relatively low level of gene activity. The locations of silver grains on the Y chromosomes were generally consistent with that revealed by the in situ hybridization using [3H]cDNA synthesized from the total Chinese hamster messenger RNA. These observations suggest that a specific subset of genomic DNA on active chromatin is the preferred site of the nick-translation.
Giant axonal neuropathy: acceleration of neurofilament transport in optic axons.
Authors: Monaco S , Autilio-Gambetti L , Zabel D , Gambetti P
Published: May 2006

Giant axonal neuropathies are a group of acquired and inherited human diseases morphologically characterized by accumulation of neurofilaments (NF) in enlargements of preterminal regions of central and peripheral axons. Slow axonal transport was studied in the optic systems of rats treated with 2,5-hexanedione, a toxic compound that produces an experimental model of giant axonal neuropathy. The transport rate of NF and of two other polypeptides of Mr 64,000 and 62,000 were selectively increased. Other components of the slow axonal transport were not affected. Acceleration of labeled NF was also observed when 2,5-hexanedione was given after [35S]methionine administration. Morphometric analysis revealed that the number of NF and the axon size were decreased in regions of optic axons proximal to the enlargements. It is suggested that acceleration of NF transport leads to a longitudinal redistribution of NF: NF decrease proximally and increase distally, forming NF-containing enlargements. Evidence was obtained that polypeptides of Mr 64,000 and 62,000 are cytoskeletal components related to intermediate filaments, normally migrating with the component a of the slow axonal transport. The 2,5-hexanedione axon may provide insight into the pathogenesis of inherited and acquired giant axonal neuropathies and offers a model to investigate the relationship between number of NF and axonal size in central axons.
Evidence that a 90-kDa phosphoprotein, an associated kinase, and a specific phosphatase are involved in the regulation of Cloudman melanoma cell proliferation by insulin.
Authors: Fleischmann R D , Pawelek J M
Published: May 2006

Proliferation of wild-type Cloudman S91 melanoma cells is inhibited when insulin is included in the culture medium. Using growth inhibition as a selective marker, we isolated variant cell lines that are either resistant to insulin or dependent upon insulin for growth. We have studied the effects of insulin on proliferation by using combined genetic and biochemical approaches. Through a series of genetic hybridization analyses, we have identified three complementation groups and determined that, in general, insulin-sensitivity is dominant to insulin-resistance. Through analyses of in vitro protein phosphorylation reactions, we have identified a protein of approximately 90 kDa (pp90) whose phosphorylation is a function of at least one of the complementation groups. Although pp90 is not phosphorylated in extracts of insulin-resistant variants, it is phosphorylated in extracts of insulin-sensitive hybrids formed between complementing resistant variants. Insulin itself exhibits little or no regulation over the phosphorylation of pp90; rather, the ability to phosphorylate pp90 correlates with the ability of cells to respond to insulin. Migration in NaDodSO4/polyacrylamide gels, solubility characteristics, and divalent cation requirements indicate that pp90 is distinct from the 95-kDa beta-subunit of the insulin receptor. Both pp90 and its associated phosphoprotein kinase are found in 30,000 X g pellets of sonicated cell lysates, whereas a specific pp90 phosphoprotein phosphatase activity is found in 30,000 X g supernatant fractions. Phosphorylation of pp90 occurs at tyrosine and serine residues. Our evidence indicates that the state of phosphorylation of pp90 is an important determinant in the regulation of cellular proliferation by insulin.
Bovine papillomavirus contains multiple transforming genes.
Authors: Yang Y C , Okayama H , Howley P M
Published: May 2006

Bovine papillomavirus type 1 (BPV-1) and its cloned full-length DNA can transform rodent cells in vitro, and the viral DNA persists as an extrachromosomal multicopy plasmid in these transformed cells. Previous studies have identified at least five discrete viral RNAs that are expressed in BPV-1 transformed cells and have shown that these transcripts share a 3' coterminus. To further define the structure of these RNAs and to characterize the functions of individual viral transcripts, we constructed a cDNA library with mRNA from BPV-1-transformed mouse C127 cells using an Okayama and Berg plasmid. From a library of 10(5) independent clones, 200 BPV-1 specific clones were isolated and characterized. Sequence analysis has revealed differential splicing patterns for the mRNA species in BPV-1 transformed cells. In conjunction with the open reading frames (ORFs) deduced from the BPV-1 DNA sequence, it is possible to predict the structure of the potential encoded proteins. The vector used to generate these cDNA clones contains mammalian cell transcriptional regulatory elements, facilitating their functional characterization. We have identified two distinct classes of cDNA clones that can each independently transform mouse C127 cells. One class of cDNA clones contains the E2 ORF intact and the second contains the E6 ORF intact. These two putative viral functions appear to act synergistically in transforming mouse C127 cells in vitro.
Ethylation of poly(dC-dG).poly(dC-dG) by ethyl methanesulfonate stimulates the activity of mammalian DNA methyltransferase in vitro.
Authors: Farrance I K , Ivarie R
Published: May 2006

Ethylation of poly(dC-dG).poly(dC-dG) with ethyl methanesulfonate (EtMes), a known carcinogen, at increasing molar ratios of EtMes/C X G base pairs progressively stimulated the methyl-accepting ability of the DNA during in vitro methylation by partially purified rat DNA (cytosine-5)-methyltransferase (EC 2.1.1.37). Maximum stimulation was 2-fold over mock-treated DNA when 2.7% of the guanines were modified at the N-7 position, the major site of ethylation by EtMes in DNA. If a CpG site "hemiethylated" at guanine N-7 mimics a hemimethylated CpG site, we calculate that the enzyme has a relative affinity for hemiethylated CpG 18-fold above unmodified CpG. If ethylation of a dioxyphosphate oxygen of the phosphodiester bond is responsible for stimulation, the relative affinity could be much higher, up to 370-fold.
Presence and source of free isopentenyladenosine in yeasts.
Authors: Laten H M , Zahareas-Doktor S
Published: May 2006

Cytokinins are a class of naturally occurring compounds that regulate growth and differentiation in tissues of higher plants. Many cytokinins are isopentenylated derivatives of adenine and its riboside, adenosine. By virtue of the post-transcriptional isopentenylation of specific anticodon loop adenosine residues in certain tRNA sequences, cytokinins are nearly universal, but tRNA-independent (de novo) cytokinin synthesis has been demonstrated in a few species. Using a radioimmunoassay, we have demonstrated that haploid strains of Saccharomyces cerevisiae and Schizosaccharomyces pombe contain, respectively, 0.8 and 0.9 microgram of the free cytokinin, N6-(delta 2-isopentenyl)adenosine, per g of cells (wet weight). Strains of both species characterized by mutations that result in deficiencies of isopentenylated tRNAs have somewhat elevated levels of free N6-(delta 2-isopentenyl)adenosine. These findings lead to the conclusion that the major, if not exclusive, source of free cytokinins in these two yeasts is a synthetic pathway independent of isopentenylated RNA turnover.
Tunicamycin blocks the incorporation of opsin into retinal rod outer segment membranes.
Authors: Fliesler S J , Basinger S F
Published: May 2006

Isolated frog retinas were incubated with radiolabeled glycoprotein precursors in the presence or absence of tunicamycin (TM), a selective inhibitor of protein N-glycosylation. In dual-label incubations, TM inhibited the incorporation of [3H]mannose into total retina Cl3CCOOH-precipitable material by 85% relative to controls, whereas incorporation of [14C]leucine was not significantly affected. In a companion single-label incubation, TM blocked the incorporation of [3H]leucine into rod outer segment (ROS) membrane Cl3CCOOH-precipitable material by 95% relative to controls. When retinas were labeled with [35S]methionine, fluorograms of NaDodSO4/polyacrylamide gels from control retinas and ROS membranes exhibited a heavily labeled component (apparent Mr approximately 37,000) which had the electrophoretic and antigenic properties of opsin, the rod visual pigment apoglycoprotein. TM-treated retinas exhibited a substantially reduced labeling of the Mr 37,000 component and incorporation of label into a component (apparent Mr approximately 32,000) not found in control retinas, which exhibited the electrophoretic and antigenic behavior of nonglycosylated opsin. ROS membranes isolated from TM-treated retinas contained neither the Mr 37,000 nor the Mr 32,000 radiolabeled species. Light-microscope autoradiograms of retinas incubated with [3H]leucine in the absence of TM exhibited bands of silver grains at the base of ROS, indicative of new membrane assembly. However, no such bands were observed in autoradiograms of TM-treated retinas. These results suggest that glycosylation of opsin is required for its incorporation into ROS membranes.
Ultrastructural features of minute chromosomes in a methotrexate-resistant mouse 3T3 cell line
Authors: Hamkalo Barbara A. , Farnham Peggy J. , Johnston Randal , Schimke Robert T.
Published: May 2006

The Miller spreading procedure was applied to mouse metaphase spreads of methotrexate-resistant 3T3 cells that contain large numbers of minute chromosomes and dihydrofolate reductase genes. There is substantial variation in both size and numbers of minutes in individual cells, the smallest of which (estimated as 5 × 10
3
kilobase pairs) would be undetected by standard light microscopic analyses. Minute chromosomes are composed of nucleosomal chromatin, which is organized into typical higher order fibers that are folded to form rosette-like structures characteristic of normal chromosome organization. There is no evidence that the DNA in minutes is linear. Minutes exist singly and in pairs, and members of a pair are connected by higher order chromatin fibers, suggesting that they are topologically interlocked. They are often closely apposed to chromosomal telomeres or arms, a configuration that may be involved in their distribution at mitosis. In addition to typical minutes, which do not possess kinetochores, a small marker chromosome possessing all of the features of a centromere region is present in parental and resistant cells. An unusual feature of this cell line is the retention of resistance, minute chromosomes, and amplified dihydrofolate reductase genes; most methotrexate-resistant mouse cell lines with minute chromosomes lose these properties when grown in the absence of methotrexate.
Neuroleptic drugs attenuate calcium influx and tension development in rabbit thoracic aorta: effects of pimozide, penfluridol, chlorpromazine, and haloperidol.
Authors: Flaim S F , Brannan M D , Swigart S C , Gleason M M , Muschek L D
Published: May 2006

This study was designed to determine whether neuroleptic drugs have calcium channel blocking activity in isolated rings of rabbit thoracic aorta. The results confirm previous findings that pimozide and penfluridol are calcium channel blockers. However, the data do not support the conclusion that these agents are selective for the voltage-sensitive calcium channel. The results also show that both haloperidol and chlorpromazine (which represent different classes of neuroleptic drugs) are also calcium channel blockers in vascular smooth muscle.