bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzymatic properties of purified Escherichia coli uvrABC proteins.
Authors: Yeung A T , Mattes W B , Oh E Y , Grossman L
Published: May 2006

The cloned uvrA and uvrB genes of Escherichia coli K-12 were amplified by linkage to the PL promoter of plasmid pKC30. The uvrC gene was amplified in the high-copy-number plasmid pRLM 24. The three gene products (purified in each case to greater than 95% purity) and ATP are required to effectively incise UV-damaged DNAs. The uvrABC proteins bind tightly to damaged sites in DNA, requiring the initial attachment of the uvrA protein in the presence of ATP before productive binding of the uvrB and uvrC proteins. Using a cloned tandem double insert of the lac p-o region as a damaged DNA substrate for the uvrABC complex and analyzing the incision both 5' and 3' to each pyrimidine dimer, we found that one break occurs 7 nucleotides 5' to a pyrimidine dimer and a second break is made 3-4 nucleotides 3' from the same pair of pyrimidines in the dimer. No such breaks are found in the strand complementary to the dimer. The size of the incised fragment in the DNA suggests that incision may be coordinated with excision reactions in repair processes.
Phorbol esters stimulate the phosphorylation of receptors for insulin and somatomedin C.
Authors: Jacobs S , Sahyoun N E , Saltiel A R , Cuatrecasas P
Published: May 2006

The effect of phorbol esters on the extent of phosphorylation of receptors for insulin and somatomedin C (insulin-like growth factor I) was studied in intact IM-9 cells that were labeled by incubation with H332PO4. The tumor-promoting phorbol esters phorbol tetradecanoate acetate (TPA) and phorbol dibutyrate, but not the inactive 4 alpha-phorbol, enhanced phosphorylation of the beta subunit of both receptors approximately 4-fold; 70 nM TPA maximally stimulated phosphorylation of both receptors, whereas concentrations less than or equal to 0.7 nM had no observable effect. Insulin also enhanced the phosphorylation of the beta subunit of the insulin receptor, and its effects appeared to be additive to those of TPA. Peptide maps indicated that at least some of the residues phosphorylated by these two agents are distinct. These results suggest a possible role of protein kinase C in regulating insulin and somatomedin C receptors.
Sarcoma growth factor from conditioned medium of virally transformed cells is composed of both type alpha and type beta transforming growth factors.
Authors: Anzano M A , Roberts A B , Smith J M , Sporn M B , De Larco J E
Published: May 2006

Sarcoma growth factor (SGF) derived from conditioned medium of Moloney sarcoma virus-transformed cells and partially purified by gel filtration (crude SGF) has been characterized by its ability both to compete with epidermal growth factor (EGF) for binding to membrane receptors and to induce anchorage-independent growth of untransformed cells. We now show that further purification of crude SGF by reverse-phase HPLC on muBondapak C18 and CN columns at pH 2 resolves it into two distinctly different polypeptides, which we call types alpha and beta transforming growth factors (TGFs). Type alpha TGF (TGF-alpha), but not type beta TGF (TGF-beta), competes for binding to the EGF receptor and induces the formation of small colonies (1,000-2,000 micron2) of normal rat kidney cells in soft agar. Both TGF-beta and EGF or TGF-alpha must be present in order to induce the formation of large colonies (7,000-15,000 micron2). Based on EGF competing equivalents as determined from a radioreceptor assay with 125I-labeled EGF in normal rat kidney cells, the relative ability of EGF and TGF-alpha to potentiate TGF-beta-dependent colony formation is in the order conditioned-medium TGF-alpha greater than EGF greater than intracellular TGF-alpha. Suboptimal concentrations of the same polypeptides give additive potentiation of the TGF-beta-dependent colony-forming response; saturating levels potentiate a similar maximum response whether used alone or in various combinations. The data indicate that the EGF-competing activity of crude SGF is due to its TGF-alpha component alone, whereas the soft-agar colony-forming activity is due to the combined action of two distinct polypeptides, TGF-alpha and TGF-beta.
Biphasic expression and function of glucose dehydrogenase in Drosophila melanogaster.
Authors: Cavener D R , MacIntyre R J
Published: May 2006

Glucose dehydrogenase (GO) was found to be expressed during the pupal stage in both sexes of Drosophila melanogaster, but is limited to the male ejaculatory duct at the adult stage. During copulation GO is transferred from males to females. Mutational analysis of the Go locus indicates that a single structural gene encodes the pupal and ejaculatory duct GO. Thus an example of an enzyme structural gene switching from non-sex-limited to sex-limited expression has been found. Go mutants are recessive lethals exhibiting a late pupal effective lethal phase. These mutants can be rescued by excising the anterior end of the pupal case 0-2 days prior to the normal adult emergence time. It appears that the function of GO in pupae is to aid in the degradation of the puparium cuticle in preparation for the eclosion of the adult.
Diffusion model of intergroup selection, with special reference to evolution of an altruistic character
Authors: Kimura Motoo
Published: May 2006

Assume a diploid species consisting of an infinite number of competing demes, each having

N
e

reproducing members and in which mating is at random. Then consider a locus at which a pair of alleles
A
and
A
′ are segregating, where
A
′ is the “altruistic allele,” which has selective disadvantage
s
′ relative to
A
with respect to individual selection, but which is beneficial for a deme in competition with other demes; namely, a deme having
A
′ with frequency
x
has the advantage
c
(
x
—
¯x
) relative to the average deme, where
c
is a positive constant and
¯x
is the average of
x
over the species. Let ϕ = ϕ(
x;t
) be the distribution function of
x
among demes in the species at time
t
. Then, we have ∂ϕ/∂
t
=
L
(ϕ) +
c
(
x
—
¯x
)ϕ, where
L
is the Kolmogorov forward differential operator commonly used in population genetics [i.e.,
L
= (1/2) (∂
2
/∂
x
2
)
V

δ
x

— (∂/∂
x
)
M

δ
x

], and
M

δ
x

and
V

δ
x

stand for the mean and variance of the change in
x
per generation within demes. As to migration, assume Wright's island model and denote by
m
the migration rate per deme per generation. By investigating the steady state, in which mutation, migration, random drift, and intra- and interdeme selection balance each other, it is shown that the index
D
=
c/m
— 4

N
e
s′

serves as a good indicator for predicting which of the two forces (i.e., group selection or individual selection) prevails; if
D
> 0, the altruistic allele predominates, but if
D
< 0, it becomes rare and cannot be established in the species.
Viruses in the etiology of atherosclerosis.
Authors: Benditt E P , Barrett T , McDougall J K
Published: May 2006

To examine the possible role of viruses in the etiology of atherosclerosis, we searched for the presence of viral genomes in arterial tissues by in situ hybridization. Because chickens infected with Marek disease virus, a herpesvirus, develop atherosclerotic lesions after infection, we looked for the presence of herpesvirus or parts thereof in human artery wall tissue, particularly in individuals with evidence of atherosclerosis. Herpesvirus probes were used on specimens of aortic wall removed from patients undergoing coronary bypass surgery. Evidence for the presence of herpes simplex viral mRNA was obtained in 13 specimens. Some of the specimens positive for herpes simplex virus appear to represent early stages in atherogenesis. Evidence for the presence of cytomegalovirus or Epstein-Barr viral genome was not observed in any of the specimens examined. We have also shown that herpes simplex virus can infect human fetal smooth muscle cells in culture. There are several ways in which viruses could operate in the pathogenesis of atherosclerosis: They could induce proliferation of artery wall intimal smooth muscle cells via injury or by genomic alterations leading to clonal expansion of intimal smooth muscle cell populations. We suggest that expression of at least a part of the herpesvirus genome in arterial smooth muscle cells may in some cases be instrumental in initiating or maintaining this enhanced cell proliferation. Furthermore, viral agents could explain other puzzling features in the occurrence of atherosclerosis and the attendant heart disease and strokes.
Ethanol acceptance as a function of genotype amounts of brain [Met]enkephalin.
Authors: Blum K , Elston S F , DeLallo L , Briggs A H , Wallace J E
Published: May 2006

Our results indicate a negative correlation between the amount of ethanol (10%) consumed and endogenous levels of brain [Met]enkephalin in C57BL/6J (alcohol-preferring) and DBA/2J (alcohol-nonpreferring) inbred mice strains. Additionally, it was found that 8 wk after 1-day starved groups of both C57BL/6J and DBA/2J mice were challenged with ethanol (10%) for 1-day acceptance, they had significantly lower levels of brain [Met]enkephalin compared with their nonalcohol-treated controls. These results suggest that the brain endogenous peptidyl opiates may play a crucial role in alcohol-seeking behavior.
Single-cell origin of mouse hemopoietic colonies expressing multiple lineages in variable combinations.
Authors: Suda T , Suda J , Ogawa M
Published: May 2006

By using a micromanipulator, single cells from blast cell colonies were individually transferred to 35-mm culture dishes for secondary colony formation. When individual colonies appeared to be mature, they were examined for cellular composition by May-Grunwald-Giemsa staining and were replated for determination of unexpressed hemopoietic potentials. We describe here a total of 50 mixed hemopoietic colonies. Seven types of colonies consisting of cells in two different lineages were seen--i.e., neutrophil-macrophage, neutrophil-eosinophil, macrophage-eosinophil, macrophage-mast cell, macrophage-megakaryocyte, macrophage-erythrocyte, and erythrocyte-megakaryocyte. Six types of colonies revealed three cell lineages--i.e., neutrophil-macrophage-eosinophil, neutrophil-macrophage-mast cell, neutrophil-macrophage-erythrocyte, macrophage-mast cell-erythrocyte, neutrophil-macrophage-megakaryocyte, and neutrophil-erythrocyte-megakaryocyte lineages. In addition, multilineage colonies expressing terminal differentiation in varying combinations of more than three lineages were present. Replating studies confirmed that the progenitors for many of these colonies are terminally committed to differentiation only in the lineages disclosed by staining. This study, thus, provides a proof for the single-cell origin of mouse hemopoietic colonies expressing various combinations of cell lineages. It also supports the hypothesis that the differentiation of multipotential hemopoietic progenitors is through progressive and stochastic restriction in cell lineages.
Alteration and activation of sequence-specific cleavage of DNA by bleomycin in the presence of the antitumor drug cis-diamminedichloroplatinum(II).
Authors: Mascharak P K , Sugiura Y , Kuwahara J , Suzuki T , Lippard S J
Published: May 2006

The antitumor drug cis-diamminedichloroplatinum(II) dramatically alters the sequence-specific cleavage of the bleomycin A2-iron(II)-O2 system. Preferred bleomycin cleavage sites adjacent to oligo(dG) regions on two restriction fragments of plasmid pBR322 DNA were masked by pretreatment with cis-diamminedichloroplatinum(II). trans-Diamminedichloroplatinum(II), which is inactive as an antitumor drug, showed similar but not identical behavior. The DNA-cleaving activity of bleomycin was substantially modified by cis-diamminedichloroplatinum(II), and a number of specific new cutting sites in guanine-rich parts of the sequence were activated by both isomers of the platinum complex. The results further emphasize the possibility that the synergism found when cis-diamminedichloroplatinum(II) and bleomycin are used in combination chemotherapy may be due to interactions at the level of DNA-drug binding.
Hemagglutinin of swine influenza virus: a single amino acid change pleiotropically affects viral antigenicity and replication.
Authors: Both G W , Shi C H , Kilbourne E D
Published: May 2006

The complete nucleotide sequence has been obtained of the H1 hemagglutinin (HA) gene of a high-yielding (H) mutant of the A/NJ/11/76(H1N1) strain of swine influenza virus in studies of a viral reassortant (X-53a) bearing this gene. This determination has permitted comparison with human influenza H1N1 prototype viruses A/WSN/33 and A/PR/8/34, with which 80% and 94% amino acid homology was found between HA1 and HA2, respectively. Partial sequences have been determined for other viral reassortants containing either H or L (low-yielding phenotype) genes derived from A/NJ/11/76. Sequence of the HA1 region of an L mutant prototype was virtually completed and differed from that of the H mutant by only four amino acid changes. Sequence analysis of four other viruses was restricted to regions of the HA with which monoclonal antibodies capable of distinguishing L and H mutants are presumed to react. Therefore, changes in these sequences are relevant to changes in viral phenotype. Change at residue 155 from Gly to Glu is associated with change from L to H HA phenotype. This site, structurally equivalent to amino acid 158 on the Wiley et al. HA model [Wiley, D. C., Wilson, I. A. & Skehel, J. J. (1981) Nature (London) 289, 373-378] is near the tip of the HA monomer adjacent to the proposed receptor binding site and therefore credibly could influence both viral antigenicity and replication. Because both L and H variants exist in nature and because revertants may be selected in the laboratory as replication variants in the absence of immunoselection, these studies provide evidence for fortuitous antigenic change in association with change in biological function, which is determined by a single base change.
Targeted and nontargeted liposomes for in vivo transfer to rat liver cells of a plasmid containing the preproinsulin I gene.
Authors: Soriano P , Dijkstra J , Legrand A , Spanjer H , Londos-Gagliardi D , Roerdink F , Scherphof G , Nicolau C
Published: May 2006

A plasmid containing the rat preproinsulin I gene was entrapped in large liposomes and intravenously administered to rats. Four hours after inoculation, the livers were processed for the isolation of hepatocytes. Kupffer cells, and endothelial cells, DNA was purified, and the exogenous DNA was detected in the different cell DNA preparations by Southern blotting. By using liposomes consisting of phospholipids and cholesterol, Kupffer cells were shown to be, on a per cell basis, the primary target for gene incorporation. In an attempt to target the liposomes to other liver cells, a glycolipid, lactosylceramide, was included in the lipid bilayer of the liposomes; this resulted in a substantial increase in the proportion of the exogenous gene in the hepatocytes and mainly in the endothelial cells, with a simultaneous decrease of this proportion in the Kupffer cells. Thus, it is shown that inclusion of a specific glycolipid within the bilayer of the liposomes may direct the DNA-containing vesicles to specific cell types in the liver.
Modification of glycogen synthase activity in isolated rat hepatocytes by tumor-promoting phorbol esters: evidence for differential regulation of glycogen synthase and phosphorylase.
Authors: Roach P J , Goldman M
Published: May 2006

Glycogen synthase (UDPglucose:glycogen 4-alpha-D-glucosyltransferase, EC 2.4.1.11), in isolated rat hepatocytes, has been identified as a novel intracellular target for tumor-promoting phorbol esters such as phorbol 12-tetradecanoate 13-acetate (TPA). Exposure of hepatocytes to TPA resulted in a 50% decrease in the activity ratio of glycogen synthase without/with glucose 6-phosphate. The inactivation was dose dependent and was half-maximal at a TPA concentration of approximately 16 nM (10 ng/ml). Phorbol and phorbol 13-monoacetate, ineffective tumor promoters, had little influence on glycogen synthase activity. Other biologically active diesters, phorbol 12,13-didecanoate, phorbol 12,13-dibutyrate, and phorbol 12,13-dibenzoate, caused significant inactivation of glycogen synthase. Glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) activity, however, was unaffected by TPA or any of the tumor-promoting phorbol esters mentioned above. It is concluded that phorbol diesters can interact in the regulatory pathway for glycogen synthase, but the lack of effect on phosphorylase argues that distinct mechanisms can operate for the control of glycogen synthase and phosphorylase.
Isolation of the yeast structural gene for the membrane-associated enzyme phosphatidylserine synthase.
Authors: Letts V A , Klig L S , Bae-Lee M , Carman G M , Henry S A
Published: May 2006

The structural gene (CHO1) for phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) was isolated by genetic complementation in Saccharomyces cerevisiae from a bank of yeast genomic DNA on a chimeric plasmid. The cloned DNA (4.0 kilobases long) was shown to represent a unique sequence in the yeast genome. The DNA sequence on an integrative plasmid was shown to recombine into the CHO1 locus, confirming its genetic identity. The cho1 yeast strain transformed with this gene on an autonomously replicating plasmid had significantly increased activity of the regulated membrane-associated enzyme phosphatidylserine synthase. Partial purification of phosphatidylserine synthase from microsomes of this transformed strain confirmed that the membrane-bound enzyme was overproduced 6- to 7-fold as compared with the wild-type strain. The strain also synthesized the product phospholipid, phosphatidylserine, at an increased rate. The transformed strain had altered proportions of a variety of other phospholipids, suggesting that their synthesis is affected by the rate of synthesis of phosphatidylserine in yeast.
Cellular calcium fluctuations in mammalian heart: direct evidence from noise analysis of aequorin signals in Purkinje fibers.
Authors: Wier W G , Kort A A , Stern M D , Lakatta E G , Marban E
Published: May 2006

Indirect evidence suggests that fluctuations, or oscillations, in the intracellular free calcium concentration [( Ca2+]i) can occur spontaneously in intact cardiac preparations, but such [Ca2+]i fluctuations have never been demonstrated directly. We used the bioluminescent Ca2+-sensitive protein aequorin to detect fluctuations in the [Ca2+]i in canine cardiac Purkinje fibers. Noise analysis of the aequorin luminescence reveals prominent peaks of power density at frequencies of 1-4 Hz; these peaks become larger and shift to higher frequencies as the [Ca2+]i increases. Caffeine and ryanodine abolish the [Ca2+]i fluctuations, suggesting that Ca2+ release and uptake by the sarcoplasmic reticulum generate these events. When [Ca2+]i fluctuations are present, less tension is produced at any given level of mean aequorin luminescence. Thus, [Ca2+]i fluctuations will undermine attempts to relate [Ca2+]i and force in intact myocardium.
Transformation-sensitive protein associated with the cell substratum of chicken embryo fibroblasts
Authors: Blenis John , Hawkes Susan P.
Published: May 2006

The interaction of cultured cells with their growth substrata has been studied as a function of oncogenic transformation by using chicken embryo fibroblasts infected with the temperature-sensitive mutant of Rous sarcoma virus, LA24, and grown in plastic culture dishes. In comparison to total cell fractions, substratum-associated material (SAM), prepared by EGTA release of transforming cells from culture dishes, is enriched in a 21-kilodalton (kDal) protein. Synthesis and deposition of this protein in SAM are stimulated within hours of transfer of cells to the permissive temperature (35°C), peak around 8 hr, and decline to levels 1.3-fold higher than those of controls at 41°C by 20 hr after the temperature shift. In contrast, incorporation of
3
H-labeled amino acids into newly synthesized fibronectin in SAM is not significantly influenced by the transformation process during this time. Furthermore, although the presence of fibronectin in SAM is influenced by cell density, the 21-kDal protein is increased in SAM of transforming cells at all densities examined. The 21-kDal protein is not present in increased amounts in SAM from normal, uninfected chicken embryo fibroblasts grown at 41°C and 35°C or from cells infected with the wild-type Rous sarcoma virus (Prague A), which are fully transformed. It is not a mannose-containing glycoprotein and does not appear to be phosphorylated. Furthermore, it is not a product of normal cell protein degradation induced by transformation but results from
de novo
protein synthesis after shift of LA24-infected cells to the permissive temperature. Finally, turnover of the 21-kDal protein is slower at 35°C than at 41°C. This amplifies the effect of increased synthesis and results in a net accumulation in SAM during the early stages of transformation.
Chimeric plasmid that replicates autonomously in both Escherichia coli and Neurospora crassa.
Authors: Hughes K , Case M E , Geever R , Vapnek D , Giles N H
Published: May 2006

A hybrid pBR322 plasmid (designated pDV1001) containing two functional Escherichia coli antibiotic resistance genes (kanr and camr) and a qa-2+ gene from Neurospora crassa transforms N. crassa qa-2- mutants to qa-2+ with a frequency of ca. 5 X 10(-5) per regenerated spheroplast (ca. 100 transformants per microgram of plasmid DNA). This plasmid can replicate autonomously without integrating into the N. crassa genome. The autonomously replicating hybrid plasmid was detected in N. crassa transformants by Southern gel hybridizations. DNA from these transformants can be recovered by retransformation back into E. coli aroD recipients and selection for chloramphenicol resistance. These E. coli transformants complement an aroD mutant. The hybrid plasmid DNA present in the E. coli transformants remains unchanged on the basis of DNA restriction enzyme analyses. The original, nonhomokaryotic N. crassa transformants can be maintained on a selective medium, but there is as yet no evidence that the self-replicating plasmid can be transmitted through meiosis. In addition, the self-replicating plasmid often integrates into the N. crassa genome and then is inherited in a generally stable fashion through meiosis. Our findings suggest that this plasmid, or some derivative of it, will prove useful as a routine shuttle vector for cloning genes in N. crassa.
cAMP-dependent protein kinase phosphorylates the nicotinic acetylcholine receptor
Authors: Huganir Richard L. , Greengard Paul
Published: May 2006

Postsynaptic membranes, rich in the nicotinic acetylcholine receptor, were isolated from the electric organ of
Torpedo californica
and shown to contain a cAMP-dependent protein kinase and a calcium/calmodulin-dependent protein kinase. The cAMP-dependent protein kinase phosphorylated the γ and δ subunits of the acetylcholine receptor. The phosphorylated subunits were identified after purification of the acetylcholine receptor by affinity chromatography on a choline carboxymethyl affinity gel. In contrast, the calcium/calmodulin-dependent protein kinase phosphorylated proteins that were separated from the acetylcholine receptor by affinity chromatography. Protein kinase inhibitor, a specific inhibitor of the catalytic subunit of cAMP-dependent protein kinase, abolished the basal endogenous phosphorylation of the γ and δ subunits of the receptor. cAMP activation of the endogenous phosphorylation of the γ and δ subunits was dose dependent with a half-maximal response at 25 nM. Studies were also carried out with acetylcholine receptor purified from
T. californica
and catalytic subunit of cAMP-dependent protein kinase purified from bovine heart. The purified acetylcholine receptor was rapidly and specifically phosphorylated on the γ and δ subunits by the purified catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 1.0 and 0.89 mol of
32
P per mol of receptor, respectively. The initial rates of phosphorylation of the γ and δ subunits of the receptor were comparable to those of histone f2B and synapsin I (protein I), two of the most effective substrates for the catalytic subunit. Under the conditions used, the γ and δ subunits had
K
m
values of 4.0 and 3.3 μM and
V
max
values of 2.7 and 2.1 μmol/min per mg, respectively. The results are consistent with the idea that the acetylcholine receptor is phosphorylated
in vivo
by a cAMP-dependent protein kinase.
Polyadenylylated nuclear RNA contains branches
Authors: Wallace John C. , Edmonds Mary
Published: May 2006

A highly charged component can be isolated from a total RNase T2 digest of nuclear polyadenylylated RNA from HeLa cells that is separable from caps by (dihydroxyboryl)aminoethyl-cellulose chromatography. Chemical and enzymatic analyses show that the component contains a 2′-5′ phosphodiester bond that creates a branch at the 2′-hydroxyl group of one nucleotide already linked to an adjoining nucleotide through the usual 3′-5′ phosphodiester bond. [Formula: see text] This structure was confirmed by analysis of a similar component isolated from nuclease P1 digests of the same nuclear polyadenylylated RNA. Branches occur in roughly 10% of nuclear polyadenylylated RNAs, including those >10S in size, but are absent from cytoplasmic polyadenylylated RNA. Possible implications for branches as intermediates in mRNA processing are discussed.
Cultured human fibroblasts synthesize and secrete thrombospondin and incorporate it into extracellular matrix.
Authors: Jaffe E A , Ruggiero J T , Leung L K , Doyle M J , McKeown-Longo P J , Mosher D F
Published: May 2006

Thrombospondin, a major glycoprotein released from alpha granules of thrombin-stimulated platelets, is a disulfide-bonded trimer of 160-kilodalton subunits. Cultured human foreskin and fetal lung fibroblasts secreted thrombospondin (determined by enzyme-linked immunosorbent assay) into the culture medium in a time-dependent manner (15.7 and 5.8 micrograms per 10(6) cells per 24 hr, respectively); secretion was blocked by cycloheximide. [3H]Thrombospondin was isolated from [3H]leucine-labeled fibroblast postculture medium and from cell layers with rabbit polyclonal or mouse monoclonal anti-thrombospondin coupled to staphylococcal protein A-Sepharose. The immunologically isolated [3H]thrombospondin migrated in NaDodSO4/polyacrylamide gels with purified marker platelet thrombospondin both with and without reduction. Immunofluorescence microscopy using rabbit polyclonal and mouse monoclonal anti-thrombospondin antibodies localized thrombospondin to the fibrillar extracellular matrix surrounding the cells. Thus, cultured human fibroblasts secrete thrombospondin and incorporate it into the extracellular matrix.
Photosystem I reaction center from the thermophilic cyanobacterium
Mastigocladus laminosus
Authors: Nechushtai R. , Muster P. , Binder A. , Liveanu V. , Nelson N.
Published: May 2006

Photosystem I reaction center was isolated from the cyanobacterium
Mastigocladus laminosus
. It contained four different subunits with molecular masses (as determined by sodium dodecyl sulfate gels) of about 70,000 (subunit I), 16,000 (subunit II), 11,000 (subunit III), and 10,000 (subunit IV) daltons. The purified reaction center contained about 100 chlorophyll a molecules per P
700
; however, they could be readily depleted down to about 50 chlorophyll a per P
700
without loss in the photochemical activities. The reaction center was active in cytochrome
c
photooxidation, but the photooxidation of an acidic cytochrome, like the
Euglena
cytochrome 552, required the presence of cations. The purified reaction center was found to be similar in several respects to the photosystem I reaction centers from higher plants and, especially, to the one isolated from green algae. Subunit I appeared on sodium dodecyl sulfate gels in the same position and possessed the same shape of an apparent double band as the corresponding subunits I of green plants and of algae. Subunits I and II of photosystem I reaction centers from
Mastigocladus
, higher plants, and green algae showed immunological crossreactivity. This observation might serve as biochemical evidence for the common evolution of the photosystem I reaction centers. In higher plants and green algae subunit II is a product of cytoplasmic ribosomes and therefore, a high degree of homology should have been preserved upon transfer of its gene from the prokaryote to the nucleus of the eukaryotes.
Polypeptide chain initiation in eukaryotes: reversibility of the ternary complex-forming reaction.
Authors: Siekierka J , Manne V , Mauser L , Ochoa S
Published: May 2006

In the last step of polypeptide chain initiation in eukaryotes, the interaction of the 40S preinitiation complex eIF-2.GTP.Met-tRNAi.40S [the complex between the 40S ribosomal subunit and the ternary complex containing equimolar amounts of eukaryotic initiation factor 2 (eIF-2), GTP, and eukaryotic initiator methionyl tRNA (Met-tRNAi)] with a 60S ribosomal subunit in the presence of mRNA, cap binding protein (with "capped" messengers), ATP, and the initiation factors eIF-3, eIF-4a, -4b, -4c, and eIF-5, results in the formation of an 80S initiation complex (Met-tRNAi.80S.mRNA) with concomitant hydrolysis of GTP and liberation of eIF-2 for recycling in subsequent initiation events. However, at physiological Mg2+ concentrations, GDP is known to have approximately equal to 100-fold greater affinity than GTP for eIF-2 and eIF-2 is believed to be released in the form of an eIF-2.GDP complex. Previously, we have shown that initiation factor SP (for eIF-2-stimulating protein) promotes the exchange of eIF-2-bound GDP for GTP and catalyzes ternary complex formation in the presence of Met-tRNAi. Binding of GDP by eIF-2 is indeed so tight that, as we now show, homogeneous preparations of eIF-2 contain upward of 0.5 mol of GDP/mol of eIF-2. We further show that, in the presence of Mg2+ and catalytic amounts of SP, ternary complex formation conforms to the overall reversible reaction eIF-2.GDP + GTP + Met-tRNAi in equilibrium eIF-2.GTP.Met-tRNAi + GDP.
Construction and characterization of cDNA clones for four respiratory syncytial viral genes.
Authors: Venkatesan S , Elango N , Chanock R M
Published: May 2006

Cytoplasmic poly(A)-containing RNA from respiratory syncytial virus-infected cells was used as a template to synthesize oligo(dT)-primed cDNAs. Discrete size classes of single-stranded cDNAs, resolved by alkali agarose gel electrophoresis, were used separately to construct double-stranded cDNAs that were subsequently inserted into the plasmid vector pBR322 at the Pst I site by means of oligo(dG)oligo(dC) tailing. After transfection of Escherichia coli, recombinant plasmids were screened mostly by serial rounds of hybrid selection of mRNAs from virus-infected cells and subsequent in vitro translation of the selected mRNAs. Comparative peptide mapping of the translation products with those of authentic virion proteins served to establish the viral origin of the cDNA recombinants. In this manner, four distinct classes of recombinant plasmids were identified. These encode sequences corresponding to those of respiratory syncytial virus nucleocapsid protein, matrix protein, phosphoprotein, and a nonstructural protein.
An
Arabidopsis thaliana
mutant defective in chloroplast dicarboxylate transport
Authors: Somerville S. C. , Ogren W. L.
Published: May 2006

Reactions of the photorespiratory pathway of C
3
plants are found in three subcellular organelles. Transport processes are, therefore, particularly important for maintaining the uninterrupted flow of carbon through this pathway. We describe here the isolation and characterization of a photorespiratory mutant of
Arabidopsis thaliana
defective in chloroplast dicarboxylate transport. Genetic analysis indicates the defect is due to a simple, recessive, nuclear mutation. Glutamine and inorganic phosphate transport are unaffected by the mutation. Thus, in contrast to previous reports for pea and spinach, glutamine uptake by
Arabidopsis
chloroplasts is mediated by a transporter distinct from the dicarboxylate transporter. Both the inviability and the disruption of amino-group metabolism of the mutant under photorespiratory conditions suggest that the primary function of the dicarboxylate transporter
in vivo
is the transfer of 2-oxoglutarate and glutamate across the chloroplast envelope in conjunction with photorespiratory nitrogen metabolism. The role commonly ascribed to this transporter, conducting malate-aspartate exchanges for the indirect export of reducing equivalents from the chloroplast, appears to be a minor one.
Hawkmoth pollination of
Mirabilis longiflora
(Nyctaginaceae)
Authors: Grant Verne , Grant Karen A.
Published: May 2006

A guild composed of very-long-tubed hawkmoth flowers (nectar tubes, 9 cm or more long), belonging to different genera and families, occurs in the American Southwest. Our knowledge of the hawkmoth associates of these flowers is fragmentary.
Mirabilis longiflora
, a member of the guild with a tube 10.0-10.5 cm long, was found to be visited and pollinated mainly by
Manduca quinquemaculata
with a proboscis 10.7-11.6 cm long in the Chiricahua Mountains of southeastern Arizona. This example fits in with four other previously reported cases. The long-tongued
Man. quinquemaculata
is now known to be associated with five species of very long-tongued hawkmoth flowers in the Southwest, and
Man. rustica
has been found on one of them.
Modifiers of free radicals inhibit in vitro the oncogenic actions of x-rays, bleomycin, and the tumor promoter 12-O-tetradecanoylphorbol 13-acetate.
Authors: Borek C , Troll W
Published: May 2006

Using short-term cultures of hamster embryo cells, we have examined the effects of the free-radical scavenger superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) and the enzyme catalase (hydrogen-peroxide:hydrogenperoxide oxidoreductase, EC 1.11.1.6) on x-ray- and bleomycin-induced transformation and on the enhancement of radiogenic transformation by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). We find that superoxide dismutase inhibits (i) transformation induced by x-ray and bleomycin and (ii) promotional action of TPA in vitro. The results suggest that the oncogenic action of x-rays and bleomycin and the enhancement of oncogenic transformation by TPA are mediated in part by free radicals. The findings also suggest that superoxide dismutase can serve as an inhibitor of oncogenesis and that its actions, as seen in this in vitro system, are most predominantly on inhibiting late events in the progression of cellular transformation--those associated with promotion.
Dynamics of pyrimidine deoxynucleoside triphosphate pools in relationship to DNA synthesis in 3T6 mouse fibroblasts.
Authors: Nicander B , Reichard P
Published: May 2006

The 3H-labeled nucleosides cytidine, deoxycytidine, and thymidine are rapidly incorporated into DNA via dCTP or dTTP pools. Between 30 and 60 min after addition of tracer amounts of a labeled nucleoside to the medium of rapidly growing 3T6 cells, dNTP pools attained a constant specific activity resulting from a steady-state equilibrium between incorporation of nucleoside, de novo synthesis, and linear incorporation of isotope into DNA. Removal of labeled deoxycytidine or thymidine depleted the dNTP pools of isotope within a few minutes and incorporation into DNA stopped. When de novo synthesis of dTTP was blocked with amethopterin, the intracellular dTTP pool rapidly reached the specific activity of thymidine of the medium and isotope incorporation into DNA then measured absolute rates of DNA synthesis. In experiments with and without amethopterin, we found no kinetic evidence for the existence of more than one dTTP pool and the decay of the pool suggested that all dTTP served as precursor of DNA. In contrast, experiments with deoxycytidine and cytidine suggested the presence of separate dCTP pools with preferential DNA synthesis from the pool labeled from cytidine.
Evolution, development, and the units of selection.
Authors: Buss L W
Published: May 2006

The "Modern Synthesis" forms the foundation of current evolutionary theory. It is based on variation among individuals within populations. Variations within individuals are believed to hold no phylogenetic significance because such variation cannot be transmitted to the germ line (i.e., Weismann's doctrine). Weismann's doctrine, however, does not apply to protists, fungi, or plants and is an entirely unsupported assumption for 19 phyla of animals. This fact requires that the Modern Synthesis be reexamined and modified.
Functional role for vasoactive intestinal polypeptide in the caudate nucleus: a 2-deoxy[14C]glucose investigation.
Authors: McCulloch J , Kelly P A , Uddman R , Edvinsson L
Published: May 2006

The quantitative autoradiographic 2-deoxy-[14C]glucose technique has been used with conscious rats to investigate the functional consequences (reflected as alterations in local rates of glucose utilization) of unilateral intrastriatal administration of vasoactive intestinal polypeptide. Intrastriatal administration of vasoactive intestinal polypeptide (20 pmol) significantly increased local glucose utilization in the injected striatum, where the increased use was localized in small punctate areas (100-500 microns wide in coronal sections) scattered throughout the nucleus at considerable distances (up to 4 mm) from the injection site. Significantly increased glucose utilization after intrastriatal injection of vasoactive intestinal polypeptide was observed in a number of regions (e.g., substantia nigra pars compacta, entopeduncular nucleus, lateral habenular nucleus, entorhinal, pyriform cortices, and amygdala) with known primary or secondary neuronal connections with the caudate nucleus. These alterations in glucose utilization were highly focal in nature, with the majority (40 of the 50 examined) of brain regions displaying unaltered rates of glucose utilization. The data provide evidence, obtained in conscious animals, that vasoactive intestinal polypeptide can modify functional processes in the caudate nucleus.
Tunicamycin inhibits ganglioside biosynthesis in neuronal cells
Authors: Guarnaccia Steven P. , Shaper Joel H. , Schnaar Ronald L.
Published: May 2006

The antibiotic tunicamycin blocks the transfer of GlcNAc-1-
P
from UDP-GlcNAc to dolichol phosphate, thereby blocking the synthesis of
N
-linked oligosaccharide chains on glycoproteins. Its effect on the biosynthesis of gangliosides has not been reported. We report that tunicamycin caused a 70-80% reduction in incorporation of [
3
H]GlcN into gangliosides and neutral glycosphingolipids of the neuroblastoma-glioma hybrid cell line NG 108-15 at antibiotic concentrations that caused a 90% reduction of the radiolabel incorporation into glycoproteins. The effect of tunicamycin on ganglioside biosynthesis was apparent after only 4 hr of incubation, and maximum inhibition was seen within 6 hr. When control or tunicamycin-treated (5 μg/ml) cells were collected and fractionated to separate glycoproteins, neutral glycosphingolipids, gangliosides, and nucleotide sugar-precursor pools, the following results were obtained: (
i
) UDP-GlcNAc and UDP-GalNAc pool sizes increased >3-fold, and specific activities decreased 50% upon treatment with tunicamycin; (
ii
) when corrected for this value, the percentage inhibition of GlcN incorporation into various glycoconjugates by tunicamycin in these cells was 82% for glycoproteins, 54% for neutral glycosphingolipids, and 50% for gangliosides; and (
iii
) the different gangliosides were affected differentially, with the most striking inhibition apparent in GM
3
biosynthesis, which was decreased 78% in the presence of tunicamycin. These data suggest that the effects of tunicamycin on glycosphingolipids as well as on glycoproteins must be considered when interpreting its effects on intact cells and organisms.
DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory.
Authors: Fischer S G , Lerman L S
Published: May 2006

DNA fragments 536 base pairs long differing by single base-pair substitutions were clearly separated in denaturing gradient gel electrophoresis. Transversions as well as transitions were detected. The correspondence between the gradient gel measurements and the sequence-specific statistical mechanical theory of melting shows that mutations affecting final gradient penetration lie within the first cooperatively melting sequence. Fragments carrying substitutions in domains melting at a higher temperature reach final gel positions indistinguishable from wild type. The gradient data and the sites of substitution bracket the boundary between the first domain and its neighboring higher-melting domain within eight base pairs or fewer, in agreement with the calculated boundary. The correspondence between the gradient displacement of the mutants and the calculated change in helix stability permits substantial inference as to the type of substitution. Excision of the lowest melting domain allows recognition of mutants in the next ranking domain.
Differential immunological crossreactivity of HeLa keratin antibodies with human epidermal keratins.
Authors: Fey S J , Larsen P M , Bravo R , Celis A , Celis J E
Published: May 2006

HeLa cells contain four keratin-like proteins having molecular weights of 50,000 (IEF 31), 48,500 (IEF 36), 44,000 (IEF 44), and 43,500 (IEF 46), respectively. Mouse polyclonal antibodies prepared against two of these keratins (IEF 31 and 46) have been used in this study to identify human epidermal keratins with common antigenic determinants. Using a sensitive immunoprecipitation procedure we show that the IEF 31 antibody crossreacts with three human acidic epidermal keratins, termed K1, K2, and K3, having molecular weights of 44,000, 47,500, and 54,000, respectively. One of these keratins (K1) comigrated with HeLa keratin IEF 44 and exhibited an identical one-dimensional peptide map. This protein is also abundant in basaliomas. In contrast to these results, the IEF 46 antibody showed no crossreactivity with any of the human acidic or basic [35S]methionine-labeled epidermal proteins. The lack of crossreactivity of this antibody was further confirmed by indirect immunofluorescence staining of cryostat sections from human split skin. These results emphasize both the similarity and diversity of antigenic determinants among HeLa and epidermal keratins.
Expression of cellular myc and mos genes in undifferentiated B cell lymphomas of Burkitt and non-Burkitt types.
Authors: Maguire R T , Robins T S , Thorgeirsson S S , Heilman C A
Published: May 2006

Burkitt lymphomas contain reciprocal translocations between chromosome 8 and one of the chromosomes containing the immunoglobulin gene loci, prompting speculation that consequent activation of a crucial gene(s) on chromosome 8 might be involved in the generation of these tumors. Recently the human counterparts of the retroviral oncogenes v-myc and v-mos have been mapped to chromosome 8. We have, therefore, analyzed the level of transcription of the cellular myc and mos genes in a variety of undifferentiated B cell lymphomas of Burkitt and non-Burkitt type that possess either an 8;14 or an 8;22 translocation. These lines expressed 2- to 5-fold more myc-specific RNA than do B cell lines without a translocation. Tumor cell lines of American origin with an 8;14 or 8;22 translocation expressed similar amounts of myc-specific RNA. Tumor cell lines of African origin contained slightly higher levels of myc-specific RNA than did those of American origin. However, level of expression does not appear to correlate with the presence or absence of Epstein-Barr virus. Therefore, a major increase in the transcription of this gene secondary to translocation is unlikely to be the cause of Burkitt lymphoma. There was no evidence of mos-related transcripts in any of the cell lines tested.
Glucocorticoid-thyroid synergism in lung maturation: a mechanism involving epithelial-mesenchymal interaction.
Authors: Smith B T , Sabry K
Published: May 2006

We studied the effects of cortisol and triiodothyronine (T3) on 20-day fetal rat lung cell cultures. Cortisol enhanced the production of surfactant-associated saturated phosphatidylcholine while T3 did not. However, T3 potentiated the cortisol effect. We observed that T3 enhanced the response of cultures enriched with alveolar type H cells to fibroblast-pneumonocyte factor (FPF). Intracellular cAMP was increased by exposure of these cultures to FPF, and T3 potentiated this increase. Unlike cortisol, T3 had no effect on production of FPF by fetal lung fibroblasts, as determined by bioassay of fractions of fibroblast-conditioned medium partially purified by column chromatography. The time course of cortisol action on mixed (fibroblast/epithelial) cultures was in keeping with the proposed mechanism: glucocorticoid induction of FPF in fibroblasts, followed by FPF induction of cAMP in epithelia and, finally, by enhanced production of saturated phosphatidylcholine. Thus, glucocorticoid acting on mesenchyme and thyroid hormone acting on epithelium have a synergistic effect on expression of differentiated epithelial function.
Macrophage activation: dissociation of cytotoxic activity from Ia-A antigen expression.
Authors: Blumenthal E J , Roberts W K , Vasil A , Talmage D W
Published: May 2006

Peritoneal macrophages were obtained from DBA/2 mice that were untreated or after the injection of bacillus Calmette-Guerin (BCG), thioglycollate broth, proteose-peptone broth, or gamma-irradiated P-815 tumor cells. These macrophages were "activated" to become cytotoxic for a fibroblast cell line (L 929) by the addition of lymphokines (LKs), lipopolysaccharide (LPS), or fibroblast interferon (IFN-beta), and the expression of I region-associated antigens (Ia-Ad) on the macrophages was examined both before and after activation. Thioglycollate-elicited macrophages became Ia-A+ when activated by LKs, but they remained Ia-A- when activated by LPS or IFN-beta. Resident macrophages and proteose-peptone-elicited macrophages remained Ia-A- when activated with LKs. Macrophages from BCG-infected mice were both Ia-A+ and cytotoxic for tumor cells without further treatment. In contrast, macrophages from mice injected with gamma-irradiated P-815 mastocytoma cells were Ia-A+ but not cytotoxic, and these macrophages could not be made cytotoxic by incubation with LKs. The cultured macrophage-like cell lines P388D1 and WEHI-3 became Ia-A+ after incubation with LKs, and this treatment amplified the cytotoxicity of both cell lines. We conclude that a number of factors are important in determining whether Ia-A expression accompanies macrophage activation and that Ia-A is irrelevant as a surface marker for macrophage activation.
Nanosecond absorption spectroscopy of hemoglobin: elementary processes in kinetic cooperativity.
Authors: Hofrichter J , Sommer J H , Henry E R , Eaton W A
Published: May 2006

A nanosecond absorption spectrometer has been used to measure the optical spectra of hemoglobin between 3 ns and 100 ms after photolysis of the CO complex. The data from a single experiment comprise a surface, defined by the time-ordered set of 50-100 spectra. Singular value decomposition is used to represent the observed spectra in terms of a minimal set of basis spectra and the time course of their amplitudes. Both CO rebinding and conformational changes are found to be multiphasic. Prior to the quaternary structural change, two relaxations are observed that are assigned to geminate recombination followed by a tertiary structural change. These relaxations are interpreted in terms of a kinetic model that points out their potential role in kinetic cooperativity. The rapid escape of CO from the heme pocket compared with the rate of rebinding observed for both R and T quaternary states shows that the quaternary structure controls the overall dissociation rate by changing the rate at which the Fe--CO bond is broken. A comparable description of the control of the overall association rates must await a more complete experimental description of the kinetics of the quaternary T state.