bluuebunny/crossref_metadata_2025 huggingface dataset:
Sodium ion modulates D
2
receptor characteristics of dopamine agonist and antagonist binding sites in striatum and retina
Authors: Makman Maynard H. , Dvorkin B. , Klein Patrice N.
Published: May 2006

Sodium ion (Na
+
) influences binding of both dopamine agonists and antagonists to D
2
receptors in striatum and retina. Also, Na
+
markedly potentiates the loss of high-affinity agonist binding due to the GTP analogue p[NH]ppG. 2-Amino-6, 7-dihydroxy-1,2,3,4-tetrahydro[5,8-
3
H]naphthalene ([
3
H]ADTN) binds exclusively to an agonist conformation of D
2
receptor in both striatum and retina, distinct from the antagonist conformation labeled by [
3
H]spiroperidol or [
3
H]domperidone in striatum or by [
3
H]spiroperidol in retina. Na
+
is not required for interaction of [
3
H]ADTN or antagonist radioligand sites with the selective D
2
agonist LY-141865, the D
2
antagonist domperidone, or nonselective dopamine agonists or antagonists; however, Na
+
is necessary for high affinity interaction of those radioligand sites with the D
2
antagonists molindone and metoclopramide. With Na
+
present, striatal sites for [
3
H]ADTN, [
3
H]spiroperidol, and [
3
H]domperidone have similar affinities for antagonists but only [
3
H]ADTN sites have high affinity for agonists. Na
+
further decreases the low affinity of dopamine agonists for [
3
H]spiroperidol binding sites. Also, Na
+
enhances [
3
H]spiroperidol and decreases [
3
H]ADTN binding. Na
+
alone causes bound [
3
H]ADTN to dissociate from at least 30% of striatal and 50% of retinal sites, and with Na
+
present [
3
H]ADTN rapidly dissociates from the remaining sites upon addition of p[NH]ppG. It is proposed that D
2
receptors in striatum and retina exist in distinct but interconvertible conformational states, with different properties depending on the presence or absence of Na
+
and of guanine nucleotide.
Mechanisms that uncouple growth and differentiation in myeloid leukemia cells: restoration of requirement for normal growth-inducing protein without restoring induction of differentiation-inducing protein.
Authors: Lotem J , Sachs L
Published: May 2006

There are different macrophage- and granulocyte-inducing (MGI) proteins. Normal myeloid precursors are induced to multiply by one form (MGI-1) and to differentiate by another form (MGI-2). There are clones of myeloid leukemia cells that no longer require MGI-1 for growth but can still be induced to differentiate by MGI-2. After induction of differentiation in these leukemia cells by adding MCI-2 or inducing endogenous production of MGI-2 by lipopolysaccharide, the differentiating leukemia cells, like normal cells, again required MGI-1 for growth. This growth requirement for MGI-1 could not be substituted for by adding other protein growth factors such as epidermal, fibroblast, or nerve growth factor or insulin. Induction of differentiation in these leukemia cells by dexamethasone, arabinonucleoside (cytosine arabinoside), or methotrexate instead of by MGI-2, did not restore the requirement of MGI-1 for growth. Mutant myeloid leukemia cells that could not be induced to differentiate by MGI-2 also did not show this restoration of the requirement of MGI-1 for growth. MGI-1 in normal cells induced cell growth and also induced MGI-2, so that the cells could then differentiate by the endogenously produced MGI-2. However, MGI-1 did not induce production of MGI-2 in the leukemia cells, even though they again required MGI-1 for growth, so that there was no induction of differentiation after adding MGI-1. This lack of induction of differentiation-inducing protein by growth-inducing protein has thus identified an effective mechanism for uncoupling of growth and differentiation in malignant cells.
Isolation and partial characterization of proteins from platelet pseudopods.
Authors: Rotman A , Makov N , Lüscher E F
Published: May 2006

Isolated pseudopods of human platelets activated by ADP, thrombin, or low temperature contain, as a major constituent, a 70,000-dalton membrane protein. This protein, which is labeled in intact platelets by [125I]iodonaphthylazide, is either pushed out of the membrane plane during activation or undergoes a conformational change or, as a result of activation, is aggregated in the pseudopod region. The relative amount of actin present in the pseudopod fraction is higher in the case of thrombin activation than that of ADP- or temperature-induced activation. The relative amounts of glycoproteins IIb and III in these pseudopod fractions were found to be similar with each of the preparative methods used and also similar to their relative concentrations in whole platelets.
Isolation of a human papillomavirus from a patient with epidermodysplasia verruciformis: presence of related viral DNA genomes in human urogenital tumors.
Authors: Green M , Brackmann K H , Sanders P R , Loewenstein P M , Freel J H , Eisinger M , Switlyk S A
Published: May 2006

The DNA genome of a human papillomavirus (HPV), tentatively designated HPV-EV, was molecularly cloned from hand to leg lesions of a patient with epidermodysplasia verruciformis, a chronic skin disease associated with a 30% risk of developing cancer. Using stringent hybridization conditions, we observed less than 5% homology between HPV-EV and the cloned genomes of HPV-1, HPV-4, HPV-5, and HPV-5a. HPV-EV DNA showed approximately 6% homology with HPV-2 and 36% homology with HPV-3. These data suggest that HPV-EV is partially related to HPV-3. Using 32P-labeled cloned HPV-EV as probe in Southern blot hybridization experiments, we detected HPV-EV-related DNA in the carcinoma in situ (Bowenoid lesion) of the vulva of the patient from which HPV-EV was isolated. HPV-EV-related DNA was detected in 2 of 10 vulva carcinomas and in 2 of 31 cervical carcinomas. Related DNA sequences were found in papillomas from each of two patients with condyloma acuminata (anogenital warts), which is of interest considering that condylomas have been reported to convert occasionally to carcinomas. The positive vulva DNAs were also probed with other cloned HPV DNAs: HPV-1, HPV-4, and HPV-5a-related sequences were not detected; HPV-3 and HPV-2 DNA probes detected strong and weak DNA bands, respectively, of the same size as found with HPV-EV. The HPV DNA sequences were present in the positive tumors mainly as free viral DNA molecules; no evidence for integration into cellular DNA was found. The emerging biological picture with papillomaviruses is that cells transformed by these viruses are maintained in a transformed state by free episomal genomes. Thus, our findings are consistent with the idea, but by no means establish, that HPVs play a role in human cancer by a similar mechanism.
Synthesis by the DNA primase of Drosophila melanogaster of a primer with a unique chain length.
Authors: Conaway R C , Lehman I R
Published: May 2006

The primase associated with the DNA polymerase alpha from embryos of Drosophila melanogaster catalyzes the synthesis of ribo-oligonucleotide primers on single-stranded M13 DNA or polydeoxythymidylate templates, which can be elongated by DNA polymerase action [Conaway, R. C. & Lehman, I. R. (1982) Proc, Natl. Acad. Sci, USA 79, 2523--2527]. The primers synthesized in a coupled primase-DNA polymerase alpha reaction with an M13 DNA template are of a unique size (15 residues); those synthesized with poly(dT) range from 8 to 15 nucleotides. Primer synthesis is initiated at multiple but nonrandom sites. Like the DNA primase of Escherichia coli and the comparable activity in intact nuclei of polyoma-infected mouse cells, the DNA primase of D. melanogaster can substitute deoxynucleotides for ribonucleotides during primer synthesis.
Differential expression of selected genes in human leukemia leukocytes.
Authors: Shiosaka T , Saunders G F
Published: May 2006

cDNA clones complementary to mRNA of cells from patients having chronic lymphocytic leukemia (CLL) were used to examine quantitative changes in the mRNA levels of specific genes in human leukemia leukocytes. Fourteen (of 400) CLL-positive clones that did not hybridize with placental mRNA were studied. Three of the 14 clones were highly represented in mRNA from CEM, a T-cell line. One clone was highly represented in mRNA from CLL and two were highly represented in mRNA from patients with chronic myelocytic leukemia. Ten clones were not significantly represented in normal leukocytes and spleen mRNA. We have identified several genes that are differentially expressed in human leukemia leukocytes.
A possible immunodominant epitope recognized by murine T lymphocytes immune to different myoglobins.
Authors: Berkower I , Buckenmeyer G K , Gurd F R , Berzofsky J A
Published: May 2006

We find that a single region on the surface of different species of myoglobin appears to be immunodominant for T lymphocytes, even though the residues in that region vary sufficiently that the T cells immune to one myoglobin do not crossreact with other myoglobins bearing substitutions at that site. Immunization of B10.S mice with sperm whale myoglobin elicits T-lymphocyte populations capable of recognizing sperm whale myoglobin but not horse myoglobin, whereas the converse is true when these mice are immunized with horse myoglobin. Using a series of myoglobin variants, we tested the effect of changes in primary sequence on the T-lymphocyte proliferative response. We were able to divide the myoglobin variants into two groups, depending on whether they cross stimulate sperm whale immune or horse immune T lymphocytes. The patterns of cross stimulation of both populations of myoglobin immune T lymphocytes were explained by amino acid substitutions at position 109. However, because sperm whale and horse myoglobin differ at this residue (glutamate vs. aspartate, respectively), T lymphocytes immune to each myoglobin do not crossreact with the other myoglobin. Additional data suggest that this immunodominant epitope also includes other residues nearby on the surface of the native molecule. Mixing experiments showed that the specificity was that of T lymphocytes and not antigen-presenting cells. Monoclonal anti-I-A blocking studies showed that both myoglobins are presented in association with the same Ia antigen. Possible explanations for the apparent immunodominance of this antigenic epitope, consisting of residue 109 and nearby residues on the surface of both myoglobins, include a peculiar immunogenicity of the surface topography of this site of a preferred orientation of the molecule imposed by antigen-presenting cells when T cells first encounter the antigen. The latter explanation is related to but distinct from "determinant selection." T-cell recognition of conformation is discussed.
Antigenic polypeptide fragments of a receptor related to the Fab fragment of human immunoglobulin from thymus-derived lymphocytes.
Authors: Marchalonis J J , Hunt J C , Maxwell J , Wang A C
Published: May 2006

Certain thymus-derived lymphocytes of man and other primates express surface components related to the variable region of immunoglobulin heavy chains but lack constant region determinants defining any known immunoglobulin class. To obtain structural information on this molecule that can be used for comparison with known immunoglobulins and other surface molecules, we isolated the T cell-derived molecule by affinity chromatography using an antiserum raised against the monomeric Fab fragment of a human Waldenström macroglobulin and subjected the isolated molecule to either proteolysis using enzymes or cleavage with cyanogen bromide, followed by isolation of polypeptides which bore the Fab- or heavy chain variable region (VH)-related antigenic markers. The intact T cell molecule had an apparent mass of 68,000 daltons and no evidence was found for covalent or noncovalent association with polypeptides resembling light chains in apparent mass. The pattern of fragments obtained by cleavage of the T cell heavy chain suggests that the molecule is comprised of domains of approximate mass 12,000 daltons. Isolation of fragments from the digests that bear Fab-related serological markers shows that the molecule can be degraded into fragments resembling the Fd and VH of standard immunoglobulin heavy chains. A procedure was developed enabling the isolation of milligram quantities of VH-related T cell products. These results support the concept of a sharing of combining site determinants between T cell receptors and immunoglobulin heavy chains and show a general formal similarity between the two classes of molecules, even though they lack shared constant region determinants. The susceptibility of the T cell molecule to be cleaved into discrete antigenic fragments by controlled proteolysis might help to explain the plethora of sizes observed for antigen-specific T cell factors.
Healthy carriers of a human retrovirus, adult T-cell leukemia virus (ATLV): demonstration by clonal culture of ATLV-carrying T cells from peripheral blood.
Authors: Gotoh Y I , Sugamura K , Hinuma Y
Published: May 2006

Adult T-cell leukemia virus (ATLV) or ATLV-associated antigen (ATLA)-positive cell clones were isolated from peripheral blood lymphocytes of all five anti-ATLA-seropositive healthy adults tested by a limiting-dilution culture method in the presence of T-cell growth factor (TCGF) but from none of six seronegative adults similarly tested. Although ATLA-positive cells were not always detected in mass cultures of the seropositive lymphocytes, they were found consistently in T-cell cloned cultures of those lymphocytes. Continuous cultures of the ATLA-positive clones obtained were dependent on TCGF. Five clones derived from each of the five donors were maintained for greater than 4 months and, during culture, all of them acquired the ability to grow without added TCGF. The ATLA-positive clones formed rosettes with erythrocytes and expressed Leu 1, Leu 3a, and Leu 4 antigens but not Leu 2a antigen. These results indicate that anti-ATLA-seropositive healthy individuals carry ATLV in T cells circulating in their peripheral blood.
Tumor-promoter-resistant cells lack trisialoganglioside response.
Authors: Srinivas L , Gindhart T D , Colburn N H
Published: May 2006

JB6 mouse epidermal cells shift irreversibly to tumor cell phenotype (anchorage independence and tumorigenicity) on treatment with phorbol esters and other tumor promoters. Exposure to phorbol 12-myristate 13-acetate (PMA) decreased the de novo synthesis of trisialoganglioside (GT) in these "promotable" JB6 cells to 5-10% of that of untreated cells. The GT decrease occurred consistently in promotion-sensitive cells and not in promotion-resistant variants. Insertion of GT into membranes of PMA-treated cells inhibited PMA promotion of transformation as measured by agar colony induction. This ability to inhibit promotion of transformation is specific to GT and is not shared by other sialoglycoconjugates, including gangliosides GM1, GD1a, and asialo-GM1. The mechanism of the blocking activity of GT must be distal to the binding of PMA to its receptors, as exogenously added GT does not inhibit specific binding of tritiated phorbol diester. GT is unable to block agar colony formation by transformed cell lines, showing that its level of action is at induction of the transformed phenotype rather than at expression of it.
Molecular cloning of a DNA sequence complementary to creatine kinase M mRNA from chickens.
Authors: Schweinfest C W , Kwiatkowski R W , Dottin R P
Published: May 2006

We have cloned and identified a DNA sequence complementary to the mRNA of creatine kinase (CK) isozyme M, although the mRNA is a minor species of the total mRNA in developing myoblasts. Poly(A)+RNA from breast and thigh muscle of 5-week-old chicks was enriched for CK mRNA by a novel procedure of sucrose gradient centrifugation in the presence of methylmercuric hydroxide. DNA complementary to this mRNA was inserted into pBR322, and colonies containing the recombinant plasmids were screened for the ability of the plasmid DNA to hybridize with and rescue CK mRNA from total muscle mRNA. Three plasmids, pCS195, pCS192, and pM35-4, could specifically rescue CK-M mRNA. CK-M mRNA was detected by in vitro translation and specific immunoprecipitation. The identity of the in vitro translation product was further confirmed by its migration in two-dimensional gels at the isoelectric point and molecular weight of CK-M. The heterogeneity of CK-M observed in vivo also was found upon translation of the CK-M mRNA which hybridizes to the plasmid.
Successful immunization with a totally synthetic diphtheria vaccine.
Authors: Audibert F , Jolivet M , Chedid L , Arnon R , Sela M
Published: May 2006

Three peptides corresponding to fragments of diphtheria toxin have been synthesized. They include the previously described tetradecapeptide and two structural analogs, the hexadecapeptide and the octadecapeptide. Conjugates of these peptides to proteins or a synthetic carrier have induced in guinea pig protection against the dermonecrotic activity of diphtheria toxin. All of the conjugates were immunogenic when administered either in complete Freund's adjuvant or with N-acetylmuramyl-L-alanyl-D-isoglutamine in aqueous medium. Positive immune response toward the octadecapeptide was obtained in mice as well. In this case, the immunogenic combinations were conjugates with bovine serum albumin administered either in Freund's adjuvant or with the muramyl dipeptide and a complete synthetic conjugate comprising both the octadecapeptide and the muramyl dipeptide covalently attached to a synthetic carrier, multichain poly(DLAla). This last immunogen, which induced the most effective immune response, is a completely synthetic immunogen with built-in adjuvanticity and induces protective antitoxic immunity when administered in a physiological medium.
NH2-terminal amino acid sequence and peptide mapping of purified human beta-lipotropin: comparison with previously proposed sequences.
Authors: Spiess J , Mount C D , Nicholson W E , Orth D N
Published: May 2006

Beta-Lipotropin was purified from human pituitary glands to a purity of greater than 90%. The amino acid compositions of beta-lipotropin and its three cyanogen bromide cleavage peptide fragments were in agreement with the structure proposed by Li and Chung [Li, C.H. & Chung, D. (1981) Int. J. Pept. Protein Res. 17, 131-142]. However, the amino acid sequence of its NH2-terminal 46 amino acid residues established here differs both from the sequence derived from the direct sequence analysis of the peptide reported by Li and Chung and from that predicted on the basis of the nucleotide sequence of the human pro-opiolipomelanocortin gene proposed by Chang et al. [Chang, A.C.Y., Cochet, M. & Cohen, S.W. (1980) Proc. Natl. Acad. Sci. USA 77,4890-4894] but agrees with the structure recently derived by direct sequence analysis by Hsi et al. [Hsi, K.L., Seidah, N.G., Lu, C.L. & Chrétien, M. (1981) Biochem. Biophys. Res. Commun. 103, 1329-1335] and predicted on the basis of nucleotide sequence analysis by Takahashi et al. [Takahashi, H., Teranishi, Y., Nakanishi, S. & Numa, S. (1981) FEBS Lett. 135, 97-102]. These discrepancies, found from residues 9 to 25 of beta-lipotropin, could result from pro-opiolipomelanocortin gene polymorphism, from the existence of multiple genes for pro-opiolipomelanocortin, or, more probably, from minor errors in nucleotide and amino acid sequence analyses.
Purine oversecretion in cultured murine lymphoma cells deficient in adenylosuccinate synthetase: genetic model for inherited hyperuricemia and gout.
Authors: Ullman B , Wormsted M A , Cohen M B , Martin D W
Published: May 2006

Alterations in several specific enzymes have been associated with increased rates of purine synthesis de novo in human and other mammalian cells. However, these recognized abnormalities in humans account for only a few percent of the clinical cases of hyperuricemia and gout. We have examined in detail the rates of purine production de novo and purine excretion by normal and by mutant (AU-100) murine lymphoma T cells (S49) 80% deficient in adenylosuccinate synthetase [IMP:L-aspartate ligase (GDP-forming), EC 6.3.4.4]. The intracellular ATP concentration of the mutant cells is slightly diminished, but their GTP is increased 50% and their IMP, four-fold. Compared to wild-type cells, the AU-100 cells excrete into the culture medium 30- to 50-fold greater amounts of purine metabolites consisting mainly of inosine. Moreover, the AU-100 cell line overproduces total purines. In an AU-100-derived cell line, AU-TG50B, deficient in adenylosuccinate synthetase and hypoxanthine/guanine phosphoribosyltransferase (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), purine nucleoside excretion is increased 50- to 100-fold, and de novo synthesis is even greater than that for AU-100 cells. The overexcretion of purine metabolites by the AU-100 cells seems to be due to the primary genetic deficiency of adenylosuccinate synthetase, a deficiency that requires the cell to increase intracellular IMP in an attempt to maintain ATP levels. As a consequence of elevated IMP pools, large amounts of inosine are secreted into the culture medium. We propose that a similar primary genetic defect may account for the excessive purine excretion in some patients with dominantly inherited hyperuricemia and gout.
Purified glucocorticoid receptor-hormone complex from rat liver cytosol binds specifically to cloned mouse mammary tumor virus long terminal repeats in vitro.
Authors: Govindan M V , Spiess E , Majors J
Published: May 2006

Purified glucocorticoid hormone-receptor complex (HRC) from rat liver binds to specific DNA sequences contained in cloned mouse mammary tumor virus (MMTV)DNA. The binding site of the hormone-receptor complex is located in the long terminal repeat (LTR) of MMTV DNA as shown by filter binding studies with labeled restriction fragments and by visualization of DNA-receptor complexes with the electron microscope. The DNAs from cloned MMTV lacking the LTR sequences were neither retained on nitrocellulose filters nor bound specifically to HRCs examined in the electron microscope. The HRC also failed to bind to restriction fragments from pBR322 and phage lambda. Specific binding of the HRC to LTR sequences is dependent upon occupancy of the receptor by a glucocorticoid. Previous work has demonstrated that the MMTV transcription is initiated within the LTR; additionally, MMTV transcription is known to be regulated by glucocorticoids. Our present results therefore support the hypothesis that HRC regulates hormone-induced transcription by binding to specific DNA sequences near the MMTV transcription start site.
Removal of O6-methylguanine from DNA by human liver fractions.
Authors: Pegg A E , Roberfroid M , von Bahr C , Foote R S , Mitra S , Bresil H , Likhachev A , Montesano R
Published: May 2006

In in vitro assays using methylated DNAs as substrates, human liver fractions were shown to be able to catalyze the removal of O6-methylguanine. The amount of removal was proportional to the amount of protein added, and the loss of O6-methylguanine occurred with stoichiometric formation of guanine in the DNA and S-methylcysteine in protein. This indicates that human liver contains a protein similar to that previously found in bacteria exposed to alkylating agents. This protein acts as a transmethylase, transferring the intact methyl group from O6-methylguanine in DNA to a cysteine residue on that protein. A similar activity is present in rodent liver, but it was found that human liver was about 10 times more active in carrying out this reaction. In contrast, there was no difference between the human and rat liver extracts in catalyzing the loss of another methylation product, 7-methylguanine, from alkylated DNA. The liver is the organ most likely to be alkylated after exposure to exogenous potential alkylating agents such as dimethylnitrosamine. The present results show that human liver has a significant capacity to repair O6-methylguanine in DNA, which has been implicated as a critical product in carcinogenesis and mutagenesis.
Aminofluorene-DNA adduct formation in Salmonella typhimurium exposed to the carcinogen N-hydroxy-2-acetylaminofluorene.
Authors: Beranek D T , White G L , Heflich R H , Beland F A
Published: May 2006

The DNA adducts formed during incubation of the hepatocarcinogen N-hydroxy-2-acetylaminofluorene with Salmonella typhimurium tester strain TA1538 were investigated to determine if the covalently bound products were identical to those adducts found in rat liver DNA and to establish the biological significance of the adducts in a mutational assay. When bacteria were exposed to N-hydroxy-2-acetylaminofluorene in the presence of a 9,000 x g supernatant from a rat liver homogenate (S9), only one adduct was detected. This adduct had chromatographic, pH-dependent partitioning, and UV spectral characteristics identical to those of N-(deoxyguanosin-8-yl)-2-aminofluorene. In the absence of S9 activation the same product was detected, but at a 85-90% lower level, which indicates that S. typhimurium also may be capable of metabolizing N-hydroxy-2-acetylaminofluorene to a reactive electrophile. When incubations were conducted with N-hydroxy-2-aminofluorene in the absence of the activation system, N-(deoxyguanosin-8-yl)-2-aminofluorene again was the major adduct. At equimolar concentrations, the arylhydroxylamine was approximately 10 times more efficient than the arylhydroxamic acid in inducing reversions in the bacteria. Comparison of the mutation rate to the level of binding in bacterial DNA gave a linear relationship with a slope of 0.96 and a correlation coefficient of 0.92. These data support previous suggestions that N-hydroxy-2-acetylaminofluorene is deacetylated by rat liver S9 to the ultimate mutagen, N-hydroxy-2-aminofluorene, and also indicate that S. typhimurium can mediate this reaction. The correlation between mutagenicity and the extent of N-(deoxyguanosin-8-yl)-2-aminofluorene adduct formation, coupled with the observation that this adduct is the major DNA adduct found in rat liver in vivo, suggests that N-(deoxyguanosin-8-yl)-2-aminofluorene may be a critical lesion for the initiation of hepatic tumorigenesis.
Molecular cloning and chromosomal mapping of a human locus related to the transforming gene of Moloney murine sarcoma virus.
Authors: Prakash K , McBride O W , Swan D C , Devare S G , Tronick S R , Aaronson S A
Published: May 2006

Human DNA was analyzed for the presence of sequences homologous to the transforming gene (v-mos) of Moloney murine sarcoma virus. A single 2.5-kilobase pair (kbp) EcoRI-generated fragment of human DNA was identified by using cloned v-mos as probe. This DNA was molecularly cloned in a bacteriophage vector. By heteroduplex and restriction enzyme analyses, this human DNA fragment, designated c-mos (human), contained a 0.65-kbp region of continuous homology with v-mos and was present as a single copy in human DNA. By testing for the presence of c-mos (human) in somatic cell hybrids possessing various numbers of human chromosomes, as well as in subclones of such hybrids, it was possible to assign c-mos (human) to human chromosome 8.
Cell cycle stage-specific accumulation of mRNAs encoding tubulin and other polypeptides in Chlamydomonas.
Authors: Ares M , Howell S H
Published: May 2006

The accumulation pattern of a number of mRNAs during the cell cycle of Chlamydomonas was examined by two-dimensional gel analysis of in vitro translation products and by RNA blot hybridization analysis. Two-dimensional gel analysis revealed that 10-15% of the 300 most abundant translation products are differentially synthesized from RNA obtained at various cell cycle stages. RNAs that direct the synthesis of alpha- and beta-tubulins and that hybridize to cloned alpha- and beta-tubulin probes accumulate coordinately during the predivision period of the cell cycle, reaching peak levels before or during division. Other RNAs represented by selected cloned cDNA probes show a number of different cell cycle patterns of accumulation. The accumulation patterns of these RNAs are not directly influenced by ongoing illumination conditions, even though alternating light-dark illumination cycles are used to synchronize Chlamydomonas cells. The results suggest that there may be a complex program of gene expression correlated with cell cycle progression in Chlamydomonas.
Erythrocyte differentiation during the metamorphic hemoglobin switch of Rana catesbeiana.
Authors: Dorn A R , Broyles R H
Published: May 2006

Anurans (frogs and toads) switch from tadpole to adult hemoglobin synthesis during metamorphosis. A number of workers have attempted to determine whether tadpole and adult Hb types are expressed in the same or different erythroid cells during the switch. If the different Hb types are found in different cells during the transition, the switch in globin gene expression occurs at an early stage of cellular differentiation. Previous studies, in which immunocytochemical techniques were used to approach this question, are in conflict in regard to the metamorphic Hb switch of the North American bullfrog Rana catesbeiana. We have purified newly differentiating erythroid cells from the blood of metamorphosing tadpoles by using Percoll gradients. These new cells have an immature morphology, are very active in the synthesis of adult Hb, and contain no detectable tadpole Hb. The tadpole cells have no detectable adult Hb, are synthetically inactive, increase in density during the switch, and are then cleared from the circulation. Thus, only adult Hb expression is detected in newly differentiating erythroid cells during metamorphosis.
Differentiation-dependent stimulation of neovascularization and endothelial cell chemotaxis by 3T3 adipocytes.
Authors: Castellot J J , Karnovsky M J , Spiegelman B M
Published: May 2006

3T3 cells that have undergone adipose differentiation in vitro secrete factor(s) that stimulate angiogenesis (neovascularization) in vivo. When medium containing 0.5% fetal calf serum was conditioned by 3T3-F442A adipocytes, it stimulated angiogenesis when placed on the chicken chorioallantoic membrane. Control medium or medium conditioned by preadipocytes did not stimulate angiogenesis, even at much higher doses. Thus, the production of the angiogenic activity is strongly dependent upon differentiation of the adipocytes. The degree of the angiogenic response to adipocyte-conditioned medium was potentiated by heparin; heparin added to unconditioned medium or to preadipocyte-conditioned medium was not angiogenic. The adipocyte-conditioned medium also strongly stimulated, in a differentiation-dependent fashion, the motility of aortic and capillary endothelial cells in a modified Boyden chamber assay. Checkerboard analysis cells indicated that 75% of the motility-stimulating activity was chemotactic in nature. The chemotactic activity has an apparent specificity for endothelial cells, in that chemotaxis of smooth muscle cells and fibroblasts was stimulated to a much lesser extent. These results, in conjunction with our previous demonstration of an endothelial cell mitogen produced by 3T3 adipocytes [Castellot, J. J., Jr., Karnovsky, M. J. & Spiegelman, B. M. (1980) Proc. Natl. Acad. Sci. USA 77, 6007-6011] indicate that the differentiation of these cells is closely linked to the production of factors that stimulate angiogenesis in vivo and growth and chemotaxis of endothelial cells in vitro.
Thymocyte rosettes: multicellular complexes of lymphocytes and bone marrow-derived stromal cells in the mouse thymus.
Authors: Kyewski B A , Rouse R V , Kaplan H S
Published: May 2006

We describe the isolation and purification of multicellular complexes composed of lymphocytes and bone marrow-derived stromal cells ("thymocyte rosettes") from the mouse thymus. These rosettes are the structural in vitro correlate of in vivo associations between lymphoblasts and I-A/E negative macrophages or medullary I-A/E positive dendritic-like cells. Both types of rosettes are preformed in vivo. The rosette-associated thymocytes display a surface antigen phenotype typical of immature thymocytes. In radiation chimeras, replacement of host thymocytes by injected bone marrow cells follows a regular pattern: donor type T cells appear first at day 11 as clusters around I-A negative macrophages and approximately 2 days later as similar clusters associated with either I-A positive cortical epithelial cells or I-A positive medullary dendritic cells. These data suggest (a) a defined sequence of lymphostromal interactions during intrathymic maturation and (b) a rapid proliferation of thymocytes after interaction with stromal cells.
Perforated postsynaptic densities: probable intermediates in synapse turnover.
Authors: Nieto-Sampedro M , Hoff S F , Cotman C W
Published: May 2006

The molecular layer of the dentate gyrus of normal rats shows a large incidence of perforated postsynaptic densities (PSDs). The perforations or discontinuities occur almost exclusively in PSDs located in spines showing a U- or W-shaped junctional profile (complex PSDs). Perforated PSDs account for 16-25% of the total complex PSD profiles in young adult rats and 12-29% of those in aged animals. The frequency of perforations in the inner molecular layer of the dentate gyrus undergoes significant changes during a cycle of nondegenerative synapse turnover induced by ipsilateral ablation of the entorhinal cortex. During the first 2 days postlesion nonperforated PSDs (simple PSDs) decrease sharply, whereas perforated PSDs change little. However, at later times (4-10 days) there is a significant increase in the number of perforated PSDs that balances the number of simple PSDs lost. Beyond 10 days postlesion the proportion of both types of PSD is restored slowly to normal--i.e., nonperforated PSDs increase in number and perforated PSDs decrease, returning to the values in unoperated animals by 120 days postlesion. This inverse relationship between small nonperforated PSDs and large perforated PSDs suggests a precursor-product relationship between them. We propose that perforated PSDs are intermediates in an ongoing cycle of synapse turnover that is a part of the normal maintenance and adaptation of the nervous system.
Differences in rates of tyrosine aminotransferase deinduction with cyclic AMP and glucocorticoids.
Authors: Lewis E J , Calie P , Wicks W D
Published: May 2006

As an indirect test of the possibility that cyclic AMP produces its stimulatory effects on the synthesis of tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) by stabilizing its mRNA, the kinetics of decline in the rate of synthesis of this enzyme was measured in rat hepatoma cells following inducer removal or addition of RNA synthesis inhibitors. In all cases in which cells were previously exposed to cyclic AMP, the decline in enzyme synthesis after removal of inducer or addition of inhibitors of RNA synthesis was 4 to 5 times more rapid than in cells exposed to a glucocorticoid (dexamethasone) which also induces the aminotransferase. Thus, it seems unlikely that cyclic AMP had been acting by stabilizing the mRNA that directs the synthesis of the aminotransferase. Possible explanations for these paradoxical results are suggested. A more rapid decline in enzyme synthesis was also seen in cells induced with both cyclic AMP and glucocorticoid, suggesting direct interaction between the two inducers.
Isolation and function of spinach leaf β-ketoacyl-[acyl-carrier-protein] synthases
Authors: Shimakata Takashi , Stumpf Paul K.
Published: May 2006

Crude spinach leaf extract readily forms the stearoyl derivative of acyl-carrier-protein (ACP) when acetyl-ACP and malonyl-ACP are incubated together. Palmitoyl-ACP is also elongated by malonyl-ACP to stearoyl-ACP. When β-ketoacyl-ACP synthase {3-oxoacyl-[ACP] synthase; acyl-[ACP]:malonyl-[ACP]
C
-acyltransferase (decarboxylating), EC 2.3.1.41} is purified with decanoyl-ACP as the assay substrate, palmitoyl-ACP elongation activity is lost. When palmitoyl-ACP is the assay substrate, another protein is isolated that specifically elongates palmitoyl-ACP to β-ketostearoyl-ACP but has no activity towards decanoyl-ACP. The first protein is designated β-ketoacyl-ACP synthase I and participates in the conversion of acetyl-ACP to palmitoyl-ACP, whereas the second protein is designated β-ketoacyl-ACP synthase II, and its substrate specificity is highly restricted to myristoyl-ACP and palmitoyl-ACP. The purification of synthase II is described, and its activity is compared to synthase I. Reconstitution experiments with the highly purified nonassociated enzymes in fatty acid synthesis plus synthases I and II clearly demonstrate the roles of these two proteins in fatty acid synthesis.
Structural variability of tRNA: small-angle x-ray scattering of the yeast tRNAphe-Escherichia coli tRNAGlu2 complex.
Authors: Nilsson L , Rigler R , Laggner P
Published: May 2006

The structure of the complex formed in solution between yeast tRNAPhe and Escherichia coli tRNAGlu2 has been studied by small-angle x-ray scattering. The complex has a radius of gyration of 4.0 nm and an electron-pair distance distribution that is incompatible with a model composed to two tRNAs joined at their complementary anticodons and exhibiting the L shape seen in the crystal. Instead a model in which the two tRNAs, still bound via the anticodons, assume a conformation with the acceptor arms folded toward the anticodon arms agrees with the observed scattering curves.
Mapping evolution with ribosome structure: intralineage constancy and interlineage variation.
Authors: Lake J A , Henderson E , Clark M W , Matheson A T
Published: May 2006

Ribosomal small subunits are organized in three general structural patterns that correspond to the eubacterial, archaebacterial, and eukaryotic lineages. Within each of these lineages, ribosomal structure is highly conserved. Small subunits from all three lineages share a common overall structure except for the following differences: (i) small subunits from archaebacteria and from the cytoplasmic component of eukaryotes both contain a feature on the head of the subunit, the archaebacterial bill, that is absent in eubacteria, and (ii) eukaryotic small subunits contain additional regions of density at the base of the subunit, the eukaryotic lobes, that are absent in archaebacteria and in eubacteria. We interpret the intralineage conservation of ribosomal three-dimensional structure as forming a phylogenetic basis for regarding archaebacteria, eubacteria, and eukaryotes as primitive lines. Although our data are separate and independent from those of Woese and Fox, they lend further support to their proposal [Woese, C. R. & Fox, G. E. (1977) Proc. Natl. Acad. Sci. USA 74, 5088-5090]. These data also provide a simple, rapid, and accurate method for classifying organisms and for identifying new lineages. Finally, interlineage variation of ribosomal structure is used to establish a rigorous framework for considering the evolution of these three lines.
Brain transglutaminase: in vitro crosslinking of human neurofilament proteins into insoluble polymers.
Authors: Selkoe D J , Abraham C , Ihara Y
Published: May 2006

The accumulation in aged human neurons of insoluble, high molecular weight filamentous polymers apparently linked by nondisulfide covalent bonds led us to examine human brain for the presence of transglutaminase (EC 2.3.2.13) and endogenous protein substrates for this crosslinking enzyme. We demonstrate the presence in brain of a transamidating enzyme that can covalently crosslink brain proteins into insoluble polymers in vitro by forming gamma-glutamyl-epsilon-lysine intermolecular bridges. Brain transglutaminase is Ca2+ dependent, has an electrophoretic mobility similar to that of erythrocyte transglutaminase, and is active in human postmortem brain from aged normal individuals and patients with Alzheimer disease (senile dementia). Brain neurofilament fractions incubated in the presence of transglutaminase, Ca2+, and the fluorescent amine dansylcadaverine form a fluorescent, nondisulfide-bonded insoluble polymer; this process is associated with a decrease in the amount of soluble neurofilament polypeptides in the preparation. Electron microscopy of the polymeric material reveals an extensive network of connecting filaments, which can be immunostained with various neurofilament antisera. Cystamine, an inhibitor of transglutaminase, prevents the neurofilament crosslinking. Glial filaments and myelin basic protein can also serve as substrates of brain transglutaminase in vitro. Although Alzheimer disease-type paired helical filaments are not formed under the specific in vitro conditions employed, the data suggest one possible mechanism for the covalent crosslinking of filaments into insoluble polymers during human neuronal aging.
lac repressor-lac operator interaction: NMR observations.
Authors: Nick H , Arndt K , Boschelli F , Jarema M A , Lillis M , Sadler J , Caruthers M , Lu P
Published: May 2006

We show here the changes in the NMR spectra of the Escherichia coli lac repressor when bound to isolated lac operator DNA. The observations focus on the aromatic residues--four tyrosines and a single histidine--in the amino-terminal DNA binding domain of the lac repressor. There is a good correlation between chemical shift changes seen by 19F NMR when compared with 1 H NMR of otherwise identical repressor--DNA complexes. The results suggest that the tyrosines do not intercalate in the DNA. The NMR spectral changes with similarly sized DNA fragments, not containing the lac operator DNA sequence, are different. Thus, the amino-terminal domain of the lac repressor is independently capable of discriminating between lac operator and nonspecific DNA sequences. There can be two amino-terminal fragments per operator in the specific complex.
Electron microscopy of complexes of isolated acetylcholine receptor, biotinyl-toxin, and avidin.
Authors: Holtzman E , Wise D , Wall J , Karlin A
Published: May 2006

The principal curarimimetic toxin of Naja naja siamensis derivatized with biotinyl groups binds specifically both to acetylcholine receptor, isolated from Torpedo californica electric tissue, and to avidin. Isolated complexes of receptor monomer or dimer, biotinyl-toxin, and avidin were negatively stained and examined in the scanning transmission electron microscope. We measured the angle made by the radius of each avidin bound at the periphery of a monomeric unit in dimer to the axis connecting the centers of the monomers, starting at the crosslink between the monomers. We infer from the distribution of these angles that one toxin binding site is located in the range of 45 degrees to 85 degrees and another at about 100 degrees further from the crosslink between the monomers. Because it is known that there are two toxin binding sites per monomer, associated with the two alpha chains, the bound avidins presumably point to portions of the alpha chains, indicating their positions relative to that portion of the delta chain located at the crosslink between monomers in dimer.
Pancreatic preproglucagon cDNA contains two glucagon-related coding sequences arranged in tandem.
Authors: Lund P K , Goodman R H , Dee P C , Habener J F
Published: May 2006

We have constructed and cloned in bacteria recombinant plasmids containing DNA complementary to the mRNA encoding a pancreatic preproglucagon (Mr 14,500), a product of cell-free translation of angler fish islet mRNAs shown previously by immunoprecipitation analyses to be a precursor of glucagon. cDNAs of 630, 180, and 120 base pairs were isolated and correspond to most of the mRNA for the preproglucagon (650 bases). The cDNAs contain a protein coding sequence of 372 nucleotides and 5'- and 3'-untranslated regions of 58 and 206 nucleotides, respectively. From the coding sequence of the cDNAs, we find that the sequence of glucagon, identical to mammalian glucagon in 20 of 29 positions, resides in the preproglucagon of 124 amino acids flanked by NH2- and COOH-peptide extensions of 52 and 43 amino acids, respectively. The peptide extensions are linked to the glucagon by Lys-Arg sequences characteristic of the sites that are cleaved during the posttranslational processing of prohormones. Notable is the finding that, following the initial Lys-Arg sequence in the COOH-peptide extension is a pentapeptide. Ser-Gly-Val-Ala-Glu, followed by another Lys-Arg and a sequence of 34 residues that shows striking homology with glucagon and the other peptides of the glucagon family--gastric inhibitory peptide, vasoactive intestinal peptide, and secretin. Thus, the preproglucagon mRNA contains two glucagon-related coding sequences arranged in tandem. The finding of Lys-Arg sequences flanking the glucagon and glucagon-related sequences suggests that these two peptides and a pentapeptide are formed in vivo by posttranslational cleavages of a common precursor.
Sequence organization and genomic distribution of the major family of interspersed repeats of mouse DNA.
Authors: Meunier-Rotival M , Soriano P , Cuny G , Strauss F , Bernardi G
Published: May 2006

We have investigated that organization and the distribution of a family of interspersed DNA repeats in the mouse genome. The repeats are at least 5600 base pairs (bp) in size and contain two contiguous BamHI endonuclease fragments, 4000 and 540 bp in size, the larger of which includes a 1350-bp EcoRI fragment studied by previous authors. The repeats are polymorphic in their restriction maps, and represent the major family of interspersed repeats in the mouse genome. The repeats are present almost exclusively in the two light major components of mouse DNA, and the base composition of their large BamHI fragments matches that of those components. The genomic distribution of the repeats is different from that of structural genes, which are present not only in the two light components but also in the two heavy components of mouse DNA. This distribution indicates that the repeats are not involved, at least in any simple way, in the regulation of gene expression.
alpha-Actinin and membrane glycoprotein IIIa are different proteins in human blood platelets.
Authors: Langer B G , Leung L L , Gonnella P A , Nachmias V T , Nachman R L , Pepe F A
Published: May 2006

It has been suggested that a platelet protein that is very similar to muscle alpha-actinin is identical to the membrane glycoprotein IIIa (GPIIIa) of platelets and is responsible for anchoring actin filaments directly into the plasma membrane of platelets. To determine if alpha-actinin and GPIIIa are related in platelets, we analyzed the purified proteins on 5% sodium dodecyl sulfate/polyacrylamide gels. The two proteins differ in mobility in both the unreduced and reduced states, and they stain differently with silver stain. In addition, alpha-actinin is a prominent component of the detergent-insoluble cytoskeletons of platelets, whereas GPIIIa is absent from these structures. By using monospecific antisera to the individual proteins, it was demonstrated that alpha-actinin and GPIIIa are immunologically distinct. We conclude that alpha-actinin and GPIIIa are different proteins in human blood platelets and that it is unlikely that alpha-actinin is an integral membrane protein.