bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of cytosolic free calcium levels by extracellular phosphate and lanthanum.
Authors: Korc M , Schöni M H
Published: May 2006

The effects of extracellular phosphate and lanthanum on cytosolic free Ca2+ [( Ca2+]i) levels were studied in isolated rat pancreatic acini. In the presence of 1.28 mM Ca2+ and 1.0 mM phosphate, the mean resting [Ca2+]i level was 120 nM. Omission of phosphate from incubation medium significantly lowered this value to 94 nM. The gastrointestinal hormone cholecystokinin octapeptide (CCK-8) rapidly enhanced both [Ca2+]i levels and 45Ca2+ efflux, irrespective of the presence or absence of phosphate. Lanthanum (0.1 mM), a compound known to block transmembrane Ca2+ fluxes, attenuated both actions of CCK-8, but only in the absence of extracellular phosphate. There was a concomitant decrease in amylase secretion induced by 0.1 nM CCK-8 but not by 10 nM CCK-8, without a significant change in cellular ATP levels. The inhibitory actions of lanthanum on CCK-8-stimulated [Ca2+]i levels were very rapid and were mimicked only by prolonged incubation of acini in Ca2+-free medium supplemented with EGTA. Omission of phosphate from incubation medium also lowered basal [Ca2+]i levels in IM-9 lymphocytes. These findings suggest that extracellular phosphate may modulate resting [Ca2+]i levels in pancreatic acini and other cell types and that mobilization of intracellular Ca2+ may partly depend on the availability of a lanthanum-sensitive pool of cell-surface Ca2+ that is not readily removed by EGTA.
Poly(ADP-ribose) may signal changing metabolic conditions to the chromatin of mammalian cells.
Authors: Loetscher P , Alvarez-Gonzalez R , Althaus F R
Published: May 2006

In mammalian cells, NAD+ serves a dual role as a respiratory coenzyme and as a substrate for the posttranslational poly(ADP-ribose) modification of chromatin proteins, catalyzed by the nuclear enzyme poly(ADP-ribose) polymerase [NAD+ ADP-ribosyltransferase, EC 2.4.2.30]. Biological evidence strongly suggests that poly(ADP-ribosyl)ation modulates chromatin functions, although the precise molecular mechanisms involved have not yet been elucidated. Here we describe conditions for the rapid uptake of exogenously supplied NAD+ by living hepatocytes in primary monolayer culture. Raising the intracellular NAD+ concentration by 70% caused a 5-fold increase of chromatin-bound poly(ADP-ribose). We conclude that the constitutive level of posttranslational poly(ADP-ribose) modifications of chromatin proteins in mammalian cells is related to the availability of NAD+, which varies in different physiological and pathological states. We propose that poly-(ADP-ribose) may serve a hitherto unrecognized function by signaling altered metabolic conditions to the chromatin and thus modulate its functions in tune with changing metabolic states.
The yeast PHO5 promoter: phosphate-control elements and sequences mediating mRNA start-site selection.
Authors: Rudolph H , Hinnen A
Published: May 2006

Transcription of PHO5 is strongly regulated in response to the level of inorganic phosphate (Pi) present in the growth medium. We have identified elements required for PHO5 expression by analyzing small deletions in the PHO5 promoter on chromosome II. The results reveal three functionally different components of the PHO5 promoter: regulatory regions, a "TATA" element, and specific mRNA initiation sites. The regulatory regions contain related 19-base-pair (bp) dyad sequences acting as phosphate-controlled upstream activation sites (UASpS). These UASpS mediate the transcriptional activation of PHO5 observed in low Pi conditions. The unlinked but coordinately regulated PHO11 promoter contains a single copy of an almost identical dyad sequence, suggesting that there is a common regulatory UASp for both genes. A TATA element is absolutely required for detectable PHO5 transcription. Specific purine-pyrimidine motifs (RRYRR) (R = purine and Y = pyrimidine) serve as PHO5 mRNA initiation sites, but only if they lie 55-110 bp downstream of a functional TATA element. Such an "initiation window" is not found in higher eukaryotes and implies mechanistic differences in the transcription machineries between yeast and higher eukaryotes.
Isolation of high-affinity memory B cells: phycoerythrin as a probe for antigen-binding cells.
Authors: Hayakawa K , Ishii R , Yamasaki K , Kishimoto T , Hardy R R
Published: May 2006

In multiparameter fluorescence-activated cell sorter studies presented here, we use the highly fluorescent protein phycoerythrin (PE) as an immunizing antigen and as a fluorescent probe to reveal and isolate antigen-binding memory B cells. We demonstrate directly that memory B cells in spleens from PE-primed mice stain brightly with PE and produce strong IgG1 anti-PE responses when sorted and cultured with L3T4+ helper T cells. That is, as few as 500 sorted PE-binding cells per culture well are sufficient to produce high-level anti-PE responses, presenting antigen in a form that induces helper T cells. Three-color immunofluorescence studies reveal that cells staining brightly with PE reside primarily in a population of B220+(IgM,IgD)- cells and comprise 0.02-0.05% of the spleen cells. Complementary studies with 2,4-dinitrophenyl memory B cells show that whereas this B220+-(IgM,IgD)- population (which comprises only 1-2% of the spleen cells) gives high-affinity anti-dinitrophenyl responses, the predominant B220+(IgM,IgD)+ population yields only low-affinity antibody. Finally, we find that although both populations have similar Ia levels, cells in the high-affinity fraction have greatly reduced levels of surface immunoglobulin.
Preservation of dry liposomes does not require retention of residual water.
Authors: Crowe J H , Spargo B J , Crowe L M
Published: May 2006

Certain sugars, particularly trehalose, dramatically alter physical properties of dry phospholipids in ways that mimic the presence of water. As a result, these sugars are capable of preserving the integrity of dry liposomes and membranes. Since these effects could conceivably be due to the presence of small amounts of water in the dry preparations of sugar and lipid, we have done careful measurements of the residual water contents in the dry samples and report the results here. Lyophilized liposomes composed of palmitoyloleoylphosphatidylcholine and phosphatidylserine (9:1) contain at most 0.2 mol of H2O per mol of lipid. When the trehalose concentration in the dry mixtures is increased, there is no increase in the apparent water content of the samples over a wide range of sugar concentrations. Over the same range of trehalose contents the maximal effect of trehalose on physical properties of the lipids and on stabilization of liposomes is achieved. We conclude that the stabilization does not require retention of residual amounts of water in the dry trehalose-phospholipid preparations. Similar studies with other sugars show a relationship between the amount of sugar interacting with the lipid and the ability of the same sugar to stabilize dry liposomes.
Selection and characterization of T-cell variants lacking molecules involved in T-cell activation (T3 T-cell receptor, T44, and T11): analysis of the functional relationship among different pathways of activation.
Authors: Moretta A , Poggi A , Olive D , Bottino C , Fortis C , Pantaleo G , Moretta L
Published: May 2006

A clone of the interleukin 2-producing Jurkat leukemia cell line termed JA3 (surface phenotype, T3+, Ti+, T44+, T11+, T40+) has been used to induce and select cell variants lacking surface molecules involved in T-cell activation. Following 200 rad of gamma-radiation (1 rad = 0.01 Gy), cells were treated with monoclonal antibodies (mAbs) directed to T3, Ti, T44, or T11 antigen and complement. After growth of the residual cells in culture, "negative" cells were cloned under limiting conditions. Depending on the specificity of the mAb used for the immunoselection, three groups of variants were obtained. (i) The use of mAbs directed to T3 or Ti resulted in cell variants that expressed the T3 Ti- T44+ Leu1+ T11+ T40+ 4F2+ HLA class I+ surface phenotype. (ii) Immunoselection with anti-T44 mAb resulted in 2 variants that shared the T3- Ti- T44- Leu1- T11+ T40+ 4F2+ HLA class I+ phenotype. (iii) Cell treatment with anti-T11 mAb resulted in 15 variants characterized by the lack of T11 antigen expression and of all the other T-cell-specific surface antigens. Therefore, it appears that the different sets of JA3 cell variants, like T cells at discrete stages of intrathymic differentiation, may follow a coordinated expression of surface differentiation antigens. Analysis of the functional responsiveness of the three distinct groups of JA3 cell variants to different stimuli showed that all produced interleukin 2 in response to A23187 calcium ionophore plus phorbol 12-myristate 13-acetate. The first group of variants (T3- Ti-) did not respond to stimulation with anti-T3, anti-Ti, or anti-T44 mAbs. Eight of 9 did not respond to phytohemagglutinin either; however, all responded to appropriate stimulatory combinations of anti-T11 mAbs (and to calcium ionophore). The second group of variants (T3-, Ti-, T44-, T11+), similar to the first group, did not respond to anti-T3, anti-Ti, anti-T44 mAbs, and phytohemagglutinin, but they were fully responsive to anti-T11 mAb. The last group of variants (lacking all the T-cell-specific surface antigens) only responded to calcium ionophore A23187.
Paradoxical anticonvulsant activity of the excitatory amino acid N-methyl-D-aspartate in the rat caudate-putamen.
Authors: Turski L , Meldrum B S , Cavalheiro E A , Calderazzo-Filho L S , Bortolotto Z A , Ikonomidou-Turski C , Turski W A
Published: May 2006

We used limbic seizures induced in rats by systemic injection of the cholinergic agonist pilocarpine (380 mg/kg; i.p.) to study the neuronal pathways within the basal ganglia that modulate seizure threshold. N-Methyl-D-aspartate (N-Me-D-Asp) is an excitatory amino acid derivative that is a powerful convulsant agent when injected into the cerebral cortex, amygdala, or hippocampus in rats. Bilateral microinjections of N-Me-D-Asp into the caudate-putamen, however, protected against limbic seizures induced by pilocarpine (injected systemically), with an ED50 of 0.7 nmol (range 0.5-1.0 nmol). Lesioning the caudate-putamen (by bilateral microinjection of the excitotoxin ibotenate) converted subconvulsant doses of pilocarpine into convulsant ones. The anticonvulsant action of N-Me-D-Asp in the caudate-putamen was reversed by blocking gamma-aminobutyrate-mediated inhibition in the substantia nigra pars reticulata or in the entopeduncular nucleus. The results suggest that the caudate-putamen and its gamma-aminobutyrate-dependent efferent pathways modulate the threshold for seizures in the limbic forebrain.
Modulation of tyrosine hydroxylase gene expression in the central nervous system visualized by in situ hybridization.
Authors: Berod A , Biguet N F , Dumas S , Bloch B , Mallet J
Published: May 2006

A rat tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] cDNA probe was used for in situ hybridization studies on histological sections through the locus coeruleus, substantia nigra, and the ventral tegmental area of the rat brain. Experimental conditions were established that yielded no background and no signal when pBR322 was used as a control probe. Using the tyrosine hydroxylase probe, we ascertained the specificity of the labeling over catecholaminergic cells by denervation experiments and comparison of the hybridization pattern with that of immunoreactivity. The use of 35S-labeled probe enabled the hybridization signal to be resolved at the cellular level. A single injection of reserpine into the rat led to an increase of the intensity of the autoradiographic signal over the locus coeruleus area, confirming an RNA gel blot analysis. The potential of in situ hybridization to analyze patterns of modulation of gene activity as a result of nervous activity is discussed.
Replication of simian virus 40 origin-containing DNA in vitro with purified proteins.
Authors: Wobbe C R , Weissbach L , Borowiec J A , Dean F B , Murakami Y , Bullock P , Hurwitz J
Published: May 2006

Simian virus 40 (SV40) DNA replication dependent on the SV40 origin of replication and the SV40 large tumor (T) antigen has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, these components included the DNA polymerase alpha-primase complex, topoisomerase I, and a fraction that contained a single-stranded DNA binding protein. The latter protein, which sediments at 5.1 S on glycerol gradients and copurifies with two major protein species of 72 and 76 kDa, was isolated solely by its ability to support SV40 DNA replication. The purified system retained the species-specific DNA polymerase alpha-primase requirement previously observed with crude fractions; the complex from HeLa cells supported SV40 replication, whereas that from calf thymus and mouse cells did not. DNA containing the polyomavirus origin of replication was replicated in a system containing polyomavirus T antigen, the HeLa single-stranded DNA binding protein-containing fraction, and DNA polymerase alpha-primase complex from mouse, but not HeLa, cells. While crude fractions yielded closed circular duplex DNA, none was detected with the purified system. Nevertheless, the addition of a crude fraction to the purified system yielded closed circular monomer products.
Virulence genes
A, G
, and
D
mediate the double-stranded border cleavage of T-DNA from the
Agrobacterium
Ti plasmid
Authors: Veluthambi K. , Jayaswal R. K. , Gelvin S. B.
Published: May 2006

Agrobacterium tumefaciens
transfers the T-DNA portion of its Ti plasmid to the nuclear genome of plant cells. Upon cocultivation of
A. tumefaciens
strain A348 with regenerating tobacco leaf protoplasts, restriction endonuclease fragments of the T-DNA were generated that are consistent with double-stranded cleavage of the T-DNA at the border sequences. The T-DNA border cleavage was also induced by acetosyringone, a compound that induces many of the virulence genes. T-DNA cleavage did not occur in
Agrobacterium
strains harboring Tn
3
-HoHo1 insertions in the
virA, -D
, or -
G
genes. Insertion mutations in
virB, -C
, or -
E
did not have any effect on the T-DNA cleavage. Complementation of the mutations in
virA, -D
, or -
G
with cosmids containing the respective wild-type genes restored the T-DNA cleavage. Since
virA
and -
G
are essential in regulating the expression of other
vir
genes in response to plant signal molecules, the
virD
gene product(s) appear to mediate double-stranded T-DNA border cleavage.
Nesting: hierarchies of allosteric interactions.
Authors: Robert C H , Decker H , Richey B , Gill S J , Wyman J
Published: May 2006

A generalization of the allosteric model is presented that incorporates a hierarchy of conformational equilibria. Such a formulation draws upon structural organization already seen in many large macromolecular systems. The functional binding properties of the macromolecule reflect conformational equilibria at each structural level. Appropriate "nested" models are used to interpret structural features and functional aspects of two hemocyanin systems with a large number (12 and 24) of binding sites.
Chlorophyll catabolism in senescing plant tissues:
In vivo
breakdown intermediates suggest different degradative pathways for
Citrus
fruit and parsley leaves
Authors: Amir-Shapira Dekel , Goldschmidt Eliezer E. , Altman Arie
Published: May 2006

High-pressure liquid chromatography was used to separate chlorophyll derivatives in acetone extracts from senescing
Citrus
fruit peel, autumnal
Melia azedarach
L. leaves, and dark-held detached parsley (
Petroselinum sativum
L.) leaves. Chlorophyllide a and another polar, dephytylated derivative accumulated in large amounts in senescing
Citrus
peel, particularly in fruit treated with ethylene. Ethylene also induced a 4-fold increase in the specific activity of
Citrus
chlorophyllase (chlorophyll chlorophyllidohydrolase, EC 3.1.1.14). Detailed kinetics based on a hexane/acetone solvent partition system showed that the
in vivo
increase in dephytylated derivatives coincided with the decrease in total chlorophyll. Polar, dephytylated derivatives accumulated also in senescing
Melia
leaves. Senescing parsley leaves revealed a very different picture. The gradual disappearance of chlorophyll a was accompanied by an increase in pheophytin a and by the transient appearance of several phytylated derivatives. Only pheophytin a and an adjacent peak were left when all the chlorophyll a had disappeared. The pathways for breakdown of chlorophyll in the
Citrus
and parsley senescence systems are discussed.
Polysialic acid units are spatially and temporally expressed in developing postnatal rat kidney.
Authors: Roth J , Taatjes D J , Bitter-Suermann D , Finne J
Published: May 2006

We report the presence of alpha(2----8)-linked polysialic acid as detected with a monoclonal antibody outside the nervous tissue in the postnatal developing rat kidney. By immunoblot analysis, the reactivity was confined to a broad band of apparent molecular mass 200-250 kDa. By immunohistochemistry, the polysialic acid units were found throughout the ureteric bud-derived collecting duct system. In developing nephrons derived from the metanephrogenic mesenchyme, polysialic acid units were only regionally and transiently expressed. Furthermore, the expression of polysialic acid units was developmentally regulated, as evidenced by their gradual disappearance concomitant with postnatal kidney maturation. These findings suggest a possible role for polysialic acid units in cell-cell contact-mediated renal differentiation processes.
Comparison of the virulent Asibi strain of yellow fever virus with the 17D vaccine strain derived from it.
Authors: Hahn C S , Dalrymple J M , Strauss J H , Rice C M
Published: May 2006

We have sequenced the virulent Asibi strain of yellow fever virus and compared this sequence to that of the 17D vaccine strain, which was derived from it. These two strains of viruses differ by more than 240 passages. We found that the two RNAs, 10,862 nucleotides long, differ at 68 nucleotide positions; these changes result in 32 amino acid differences. Overall, this corresponds to 0.63% nucleotide sequence divergence, and the changes are scattered throughout the genome. The overall divergence at the level of amino acid substitution is 0.94%, but these changes are not randomly distributed among the virus protein. The capsid protein is unchanged, while proteins NS1, NS3, and NS5 contain 0.5% amino acid substitutions, and proteins ns4a and ns4b average 0.8% substitutions. In contrast, proteins ns2a and ns2b have 3.0 and 2.3% amino acid divergence, respectively. The envelope protein also has a relatively high rate of amino acid change of 2.4% (a total of 12 amino acid substitutions). The large number of changes in ns2a and ns2b, which are largely conservative in nature, may result from lowered selective pressure against alteration in this region; among flaviviruses, these polypeptides are much less highly conserved than NS1, NS3, and NS5. However, many of the amino acid substitutions in the E protein are not conservative. It seems likely that at least some of the difference in virulence between the two strains of yellow fever virus results from changes in the envelope protein that affect virus binding to host receptors. Such differences in receptor binding could result in the reduced neurotropism and vicerotropism exhibited by the vaccine strain.
Progressive dysplasia and aneuploidy are hallmarks of mouse skin papillomas: relevance to malignancy.
Authors: Aldaz C M , Conti C J , Klein-Szanto A J , Slaga T J
Published: May 2006

We report a systematic histopathologic study of papillomas at different times during promotion, correlating the results with those from cytogenetic analysis of the same tumors. Papillomas were induced in SENCAR mice by two-stage carcinogenesis (7,12-dimethylbenz[a]anthracene and phorbol 12-myristate 13-acetate). Individual tumors were randomly sampled at different times during promotion, and histopathologic and cytogenetic studies were carried out on every tumor. Early during promotion (10 weeks), most papillomas were well-differentiated hyperplastic lesions with mild or no cellular atypia. No tumors showed severe dysplastic changes. By 20 weeks of promotion, a dramatic drop had occurred in the number of lesions with no dysplasia. Most of the tumors presented moderate dysplasia, and some already showed severe dysplastic changes. At later stages (30-40 weeks), most of the papillomas were classified as moderately or severely dysplastic papillomas, and several were considered to be intrapapillomatous carcinomas. This histopathologic evaluation was supported by nuclear measurements performed on papillomas at different time points. Chromosomal abnormalities followed a similar trend. Papillomas seem to start as diploid lesions, but between 10 and 20 weeks of promotion, hyperdiploid cells can be observed in almost every tumor. In some cases the stem line was taken over by aneuploid clones. At 40 weeks of promotion, all papillomas were aneuploid, most of them with hyperdiploid stem lines. A positive correlation was found between the histological and cytogenetic studies, with the most aggressive and atypical tumors being the more aneuploid. These results support the idea that most, if not all, papillomas are truly premalignant lesions in different stages of the potential progression toward malignancy. Chromosomal abnormalities might play an important role in the sequence of events leading to malignancy.
A receptor-binding region in human choriogonadotropin/lutropin beta subunit.
Authors: Keutmann H T , Charlesworth M C , Mason K A , Ostrea T , Johnson L , Ryan R J
Published: May 2006

Synthetic fragments have not been widely used thus far to evaluate structure-activity relations in the glycoprotein hormones. We prepared a series of peptides representing the intercysteine "loop" sequence (residues 38-57) in human choriogonadotropin (hCG) and lutropin (hLH) beta subunits, anticipating that it might be oriented toward the surface and accessible to receptors. The peptides were characterized chemically and tested for bioactivity by binding to rat ovarian membrane receptor and stimulation of Leydig cell testosterone production. The hCG beta-(38-57) and hLH beta-(38-57) peptides inhibited binding of 125I-labeled hCG half-maximally at 1.51 X 10(-4) and 2.03 X 10(-5) M, respectively, while other peptide hormones and fragments from elsewhere in the beta subunit were inactive. Both peptides stimulated testosterone production, with half-maximal responses at 3.55 X 10(-5) M (hCG) and 2.18 X 10(-5) M (hLH). By radioimmunoassay with an antibody to thyroglobulin-conjugated hCG beta-(38-57) peptide, native hCG and beta subunit were highly reactive, as were the reduced and carboxymethylated subunit and peptide. Helical-wheel projection predicted an amphipathic region in the N-terminal portion of the 38-57 sequence, and circular dichroic measurements showed an increase in ordered structure, especially alpha-helix, when the 38-57 peptides were transferred from an aqueous to a more lipophilic (90% trifluoroethanol) environment. These results indicate that the 38-57 region of beta subunit is exposed on the surface and constitutes a component in the receptor-binding domain for hCG and hLH. A region of amphipathic-helical structure in the 38-57 sequence may promote hormone-receptor interactions in a manner proposed for several other peptide hormones.
Role of an adenovirus E2 promoter binding factor in E1A-mediated coordinate gene control.
Authors: Kovesdi I , Reichel R , Nevins J R
Published: May 2006

A product of the adenovirus gene E1A is responsible for the stimulation of transcription from six viral promoters as well as at least two cellular promoters. We have detected a HeLa cell factor, termed E2 promoter binding factor (E2F), that appears to mediate the transcriptional stimulation of the viral E2 promoter. Competition experiments revealed that E2F did not recognize and bind to the E1B, E3, E4, or major late promoter sequences. Furthermore, three additional promoters stimulated by E1A, heat shock protein 70, beta-globin, and early simian virus 40, do not bind E2F. In contrast, the factor does recognize sequences in the E1A enhancer, and within the E1A enhancer are duplicated binding sites for E2F. Finally, a single E2F binding site from the E1A enhancer can confer increased transcription to a mouse beta-globin promoter, dependent on the action of the E1A gene product. This stimulation requires binding of E2F since methylation of the binding site, which blocks binding in vitro, reduces transcription stimulation in vivo. We, therefore, conclude that E2F is likely to be responsible for the E1A-mediated stimulation of the E1A gene as well as the E2 gene but is not involved in the activation of the other E1A-inducible promoters.
Insulin inhibition of hepatic cAMP-dependent protein kinase: decreased affinity of protein kinase for cAMP and possible differential regulation of intrachain sites 1 and 2.
Authors: Gabbay R A , Lardy H A
Published: May 2006

In hepatocytes stimulated with 8-bromo-cAMP, insulin decreases the affinity of the cAMP-dependent protein kinase for cAMP, shifting the Ka without affecting the Vmax activity. This occurs under conditions where cyclic adenine nucleotide concentrations are unchanged. We report here that glycogenolysis stimulated by 8-(4-chlorophenylthio)-cAMP, an analog with 100 times tighter affinity than cAMP for the protein kinase regulatory subunit, was only slightly antagonized by insulin. The tight binding of this analog appears to overcome the protein kinase affinity change induced by insulin. The relative importance of the two intrachain cAMP binding sites of the cAMP-dependent protein kinase regulatory subunit was investigated by using analogs with relative selectivity for each site. Analogs exhibiting preferential binding to site 2 were far less sensitive to insulin antagonism than were analogs binding preferentially at site 1 and less well at site 2. No other property of these analogs, including the rate of hydrolysis by phosphodiesterase, the IC50 for phosphodiesterase, the Ka for protein kinase, or the type I versus type II kinase specificity, could account for the ability of insulin to antagonize glycogenolysis stimulated by these analogs. These data indicate that insulin may act to decrease the affinity of protein kinases for cAMP through a possible regulation of intrachain site 2 binding.
Activation of pp60c-src protein kinase activity in human colon carcinoma.
Authors: Bolen J B , Veillette A , Schwartz A M , DeSeau V , Rosen N
Published: May 2006

The tyrosine-specific protein kinase activity of pp60c-src molecules obtained from human colon carcinoma tissues and tumor-derived cell lines was found to be elevated over that from normal colon tissues or cultures of normal colon mucosal cells. The elevated pp60c-src protein kinase activity in tumor tissues and in cultured colon carcinoma cells does not appear to result solely from an increase in the abundance of the c-src-encoded protein, suggesting that the specific activity of the pp60c-src tyrosine phosphotransferase is enhanced. These results raise the possibility that activation of the pp60c-src protein kinase may contribute to the genesis of human colon tumors.
Stimulation of mast cells leads to cholesterol accumulation in macrophages in vitro by a mast cell granule-mediated uptake of low density lipoprotein.
Authors: Kokkonen J O , Kovanen P T
Published: May 2006

The uptake of low density lipoprotein (LDL) by cultured mouse macrophages was markedly promoted by isolated rat mast cell granules present in the culture medium. The granule-mediated uptake of LDL enhanced the rate of cholesteryl ester synthesis in the macrophages, the result being accumulation of cholesteryl esters in these cells. Binding of LDL to the granules was essential for the granule-mediated uptake of LDL by macrophages, for the uptake process was prevented by treating the granules with avidin or protamine chloride or by treating LDL with 1,2-cyclohexanedione, all of which inhibit the binding of LDL to the granules. Inhibition of granule phagocytosis by the macrophages with cytochalasin B also abolished the granule-mediated uptake of LDL. Finally, mouse macrophage monolayers and LDL were incubated in the presence of isolated rat serosal mast cells. Stimulation of the mast cells with compound 48/80, a degranulating agent, resulted in dose-dependent release of secretory granules from the mast cells and a parallel increase in cholesteryl ester synthesis in the macrophages. The results show that, in this in vitro model, the sequence of events leading to accumulation of cholesteryl esters in macrophages involves initial stimulation of mast cells, subsequent release of their secretory granules, binding of LDL to the exocytosed granules, and, finally, phagocytosis of the LDL-containing granules by macrophages.
Lentropin, a protein that controls lens fiber formation, is related functionally and immunologically to the insulin-like growth factors.
Authors: Beebe D C , Silver M H , Belcher K S , Van Wyk J J , Svoboda M E , Zelenka P S
Published: May 2006

Lentropin, a factor present in the vitreous humor of the eye, stimulates lens fiber differentiation from chicken embryo lens epithelial cells in vitro. Lentropin has been partially purified but has not been isolated in sufficient quantity or purity for direct comparison with other growth and differentiation factors. Previous studies have shown that insulin and fetal bovine serum share with lentropin the ability to stimulate lens fiber formation from cultured epithelial cells. In the present study, a number of hormones and growth factors were assayed for lentropin activity. Of those tested, the only substances that had this activity were the insulin-like growth factors (IGFs) somatomedin C (Sm-C/IGF-I) and multiplication-stimulating activity (MSA/IGF-II). Sm-C/IGF-I was approximately 30 times more potent than insulin or MSA/IGF-II in promoting fiber cell formation. A monoclonal antibody to human Sm-C/IGF-I inhibited purified Sm-C/IGF-I, fetal bovine serum, and chicken vitreous humor from stimulating fiber cell differentiation in vitro. This antibody has been shown not to crossreact with insulin and did not block insulin-stimulated lens fiber formation. These findings indicate that lentropin is related to the IGFs and that these factors may play important roles in controlling cell differentiation, in addition to their better-known ability to stimulate cell division.
Loss of heterozygosity in human ductal breast tumors indicates a recessive mutation on chromosome 13.
Authors: Lundberg C , Skoog L , Cavenee W K , Nordenskjöld M
Published: May 2006

The genotypes at chromosomal loci defined by recombinant DNA probes revealing restriction fragment length polymorphisms were determined in constitutional and tumor tissue from 10 cases of ductal breast cancer: eight premenopausal females and two males. Somatic loss of constitutional heterozygosity was observed at loci on chromosome 13 in primary tumor tissue from three females and one male. In two cases, specific loss of heterozygosity at three distinct genetic loci along the length of the chromosome was observed. In another case, concurrent loss of alleles at loci on chromosomes 2, 13, 14, and 20 was detected, whereas a fourth case showed loss of heterozygosity for chromosomes 5 and 13. In each instance, the data were consistent with loss of one of the homologous chromosomes by mitotic nondisjunction. Analysis of loci on several other chromosomes showed retention of constitutional heterozygosity suggesting the relative specificity of the events. In contrast, similar analyses of other breast cancers, including comedocarcinoma, medullary carcinoma, and juvenile secretory carcinoma, showed no loss of alleles at loci on chromosome 13. These data indicate that the pathogenesis of ductal breast cancer may, in a substantial proportion of cases, involve unmasking of a recessive locus on chromosome 13 and suggest the involvement of such a locus in heritable forms of this disease.
Mechanism of the t(14;18) chromosomal translocation: structural analysis of both derivative 14 and 18 reciprocal partners.
Authors: Bakhshi A , Wright J J , Graninger W , Seto M , Owens J , Cossman J , Jensen J P , Goldman P , Korsmeyer S J
Published: May 2006

To elucidate the mechanism of the t(14;18)(q32;q21) chromosomal translocation found in follicular lymphoma, we examined the structure of both derivative (der) chromosomal breakpoints as well as their germ-line predecessors. We noted that chromosome segment 18q21 was juxtaposed with immunoglobulin heavy (H) chain gene diversity (DH) regions on all five der(18) chromosomes we examined, and we confirmed the juncture with immunoglobulin H-chain gene joining (JH) regions on the der(14) chromosome. However, the t(14;18) was not fully reciprocal in that chromosome 14 DNA between the DH and JH regions was deleted. Furthermore, extra nucleotides, reminiscent of "N" segments, were present at the der(14) and possibly der(18) junctions. This indicates that despite the mature B-cell phenotype of follicular lymphoma, the t(14;18) occurs during attempted DH-JH joining, the earliest event in immunoglobulin rearrangement in a pre-B-cell. Our detailed analysis of the germ-line 18q21 region indicated that most breakpoints clustered within a 150-base-pair major breakpoint region. However, we found no evidence for evolutionarily conserved immunoglobulin-like recombinational signals at 18q21, arguing against a role for immunoglobulin recombinase in chromosome 18 breakage. Instead, a direct repeat duplication of chromosome 18 sequences was discovered at both chromosomal junctures, typical of the repair of a naturally occurring staggered double-stranded DNA break. These results prompt a translocation model with illegitimate pairing of a staggered double-stranded DNA break at 18q21 and an immunoglobulin endonuclease-mediated break at 14q32 and with N-segment addition, repair, and ligation to generate der(14) and der(18) chromosomes.
Metallothionein genes in Drosophila melanogaster constitute a dual system.
Authors: Mokdad R , Debec A , Wegnez M
Published: May 2006

We have selected a metallothionein (MT) cDNA clone from a cadmium-resistant Drosophila melanogaster cell line. This clone includes an open reading frame coding for a 43-amino acid protein whose characteristics are a high cysteine content (12 cysteines, 28% of all residues) and a lack of aromatic amino acids. This protein differs markedly from the Drosophila MT (Mtn gene) previously reported [Lastowski-Perry, D., Otto, E. & Maroni, G. (1985) J. Biol. Chem. 260, 1527-1530). The MT system of Drosophila thus consists of at least two distantly related genes, in sharp contrast with vertebrate MT systems, in which the different members of MT gene families display high similarity. The gene corresponding to our MT cDNA (Mto) is inducible in Drosophila cell lines and in both larval and adult flies.
Alternative processing of bovine growth hormone mRNA: nonsplicing of the final intron predicts a high molecular weight variant of bovine growth hormone.
Authors: Hampson R K , Rottman F M
Published: May 2006

We have detected a variant species of bovine growth hormone mRNA in bovine pituitary tissue and in a stably transfected bovine growth hormone-producing cell line. Analysis of this variant mRNA indicated that the last intervening sequence (intron D) had not been removed by splicing. Inspection of the sequence of intron D reveals an open reading frame through the entire intron, with a termination codon encountered 50 nucleotides into the fifth exon, which is shifted from the normal reading frame in this variant mRNA. If translated, this variant mRNA would encode a growth hormone-related polypeptide having 125 amino-terminal amino acids identical to wild-type growth hormone, followed by 108 carboxyl-terminal amino acids encoded by the 274 bases of intron D along with the first 50 nucleotides of exon 5. This variant polypeptide would be 42 amino acids longer than wild-type bovine growth hormone or approximately 5000 greater in molecular weight. The intron D-containing variant of bovine growth hormone mRNA was demonstrated to exist on polysomes, suggesting that this mRNA species is translated into a polypeptide. Cytosolic mRNA species containing any of the other three introns of the bovine growth hormone gene were not detectable.
Relocation of a protease-like gene segment between two retroviruses.
Authors: McClure M A , Johnson M S , Doolittle R F
Published: May 2006

An anomalous sequence in certain lentiviruses was found to be related to a region in a completely different part of the simian retrovirus type I (SRV-I) and its close relative, the hamster intracisternal A particle (IAP-H18). The segment is not present in the human immunodeficiency virus (HIV), which is also a lentivirus, nor is it found in any one of a dozen other retroviruses whose sequences have been reported. These observations imply that a horizontal transfer of newly acquired genetic information has taken place between an SRV-I-type virus and one of the lentivirus type, and that this event occurred more recently than did the divergence of members of this latter group and HIV. Comparison of the viral nucleic acid sequences that encode these segments revealed the presence of imperfect direct nucleotide repeats resembling the retroviral endonuclease cleavage sites at the 5' and 3' ends of these regions.
Neural networks that learn temporal sequences by selection.
Authors: Dehaene S , Changeux J P , Nadal J P
Published: May 2006

A model for formal neural networks that learn temporal sequences by selection is proposed on the basis of observations on the acquisition of song by birds, on sequence-detecting neurons, and on allosteric receptors. The model relies on hypothetical elementary devices made up of three neurons, the synaptic triads, which yield short-term modification of synaptic efficacy through heterosynaptic interactions, and on a local Hebbian learning rule. The functional units postulated are mutually inhibiting clusters of synergic neurons and bundles of synapses. Networks formalized on this basis display capacities for passive recognition and for production of temporal sequences that may include repetitions. Introduction of the learning rule leads to the differentiation of sequence-detecting neurons and to the stabilization of ongoing temporal sequences. A network architecture composed of three layers of neuronal clusters is shown to exhibit active recognition and learning of time sequences by selection: the network spontaneously produces prerepresentations that are selected according to their resonance with the input percepts. Predictions of the model are discussed.
A maternal mRNA localized to the animal pole of Xenopus eggs encodes a subunit of mitochondrial ATPase.
Authors: Weeks D L , Melton D A
Published: May 2006

We have previously isolated several cDNA clones of mRNAs that have the unusual property of being localized to either the animal pole or the vegetal pole of frog eggs. To gain insight into the function of these maternal mRNAs we have determined their DNA sequence and deduced the sequence of the proteins they encode. Here we report that An2, an mRNA localized to the animal pole of Xenopus oocytes and eggs, codes for the alpha chain of mitochondrial ATPase. Furthermore, we compare the intracellular localization of the An2 mRNA and mitochondria in oocytes and eggs and find that they do not have the same degree of localization. In the light of these results we discuss possible reasons for the maternal localization of the An2 mRNA.
Temperature-sensitive mutations in the yeast DNA polymerase I gene.
Authors: Budd M , Campbell J L
Published: May 2006

Seven mutations that yield thermolabile DNA polymerases have been isolated in the DNA polymerase I gene, POL1, of Saccharomyces cerevisiae. Strains carrying the mutant genes are temperature sensitive for growth. The pol1 mutants were identified by a method that has general applicability for identification of both temperature-sensitive and null mutations. A plasmid containing a mutagenized pol1 gene was transformed into a strain in which the only functional copy of the POL1 gene was carried on an unstable plasmid. The genes conferring temperature-sensitive growth were detected after elimination of the unstable plasmid containing the wild-type gene. DNA polymerase I isolated from each of the mutants is defective at both 23 degrees C and 36 degrees C. DNA synthesis is deficient in vivo at 36 degrees C in all the mutants, while RNA synthesis is normal in all but one of the mutants. The terminal phenotype of pol1 temperature-sensitive mutants is dumbbell-shaped cells in which the nucleus has migrated to, but apparently not entered, the isthmus separating the mother and the daughter. The POL1 gene is located on chromosome XIV approximately 2 centimorgans away from met4.
High-frequency expression of a conserved kappa light-chain variable-region gene in chronic lymphocytic leukemia.
Authors: Kipps T J , Fong S , Tomhave E , Chen P P , Goldfien R D , Carson D A
Published: May 2006

Malignant B lymphocytes from several patients with chronic lymphocytic leukemia (CLL) were examined for reactivity with murine monoclonal antibody 17.109. This antibody, prepared against the rheumatoid factor (RF) paraprotein Sie, recognizes a crossreactive idiotype on 48% of human IgM RF paraproteins, but does not react with IgM paraproteins without RF activity or substantially with normal pooled immunoglobulin. The 17.109-reactive idiotype is a marker for a kappa III variable-region gene, designated V kappa RF, that is conserved in outbred human populations. In a limited study of 31 CLL patients, the leukemic cells from 5 of 20 patients with kappa light chain-expressing CLL were recognized by the 17.109 monoclonal antibody. Despite having malignant cells specifically reactive with this antibody, patients with 17.109-positive CLL did not have elevated serum levels of circulating antibody bearing 17.109-reactive determinants. Total RNAs isolated from the CLL B lymphocytes, or from hybridomas produced by fusing the CLL cells with the WI-L2-729-HF2 cell line, were fractionated electrophoretically and examined by blot hybridization. Under stringent hybridization conditions capable of discerning a single base-pair mismatch, RNA from the 17.109-idiotype-positive CLL cells hybridized to synthetic oligonucleotide probes corresponding to framework and complementary-determining regions in the V kappa RF gene. The high frequency of the 17.109-associated idiotype and the V kappa RF gene in CLL suggests that the disease may arise from B lymphocytes that express a restricted set of inherited immunoglobulin variable-region genes with little or no somatic mutation.
Preprocholecystokinin processing in the normal human anterior pituitary.
Authors: Rehfeld J F
Published: May 2006

The processing of preprocholecystokinin in human pituitary extracts was investigated using gel and ion-exchange chromatography monitored by sequence-specific radioimmunoassays before and after incubation with trypsin, carboxypeptidase B, and arylsulfatase. Whereas the neural lobe contained only the bioactive alpha-carboxyamidated cholecystokinin (CCK) peptides (32 pmol/g), of which CCK-8 predominated, the anterior lobe contained substantial amounts of three large nonamidated procholecystokinin fragments (95 pmol/g; Mrs, 9000, 7000, and 5000) and small amounts of alpha-amidated CCK (8.3 pmol/g). The latter occurred only in the following large molecular forms: component I, CCK-58, and traces of CCK-33. Corticotrophic tumors processed the large forms to small CCK-8-like forms as are found in the brain and in the gut. The results show that a hormone gene, although translated, is expressed only to a limited extent as mature, active peptide outside the principal production region(s). Thus the processing of CCK to small alpha-amidated peptides in the less-differentiated tumor tissue supports the hypothesis that differentiation of endocrine cells may be sustained also at the posttranslational level.
A point mutation in the tobacco mosaic virus capsid protein gene induces hypersensitivity in
Nicotiana sylvestris
Authors: Knorr David A. , Dawson William O.
Published: May 2006

In
Nicotiana sylvestris
, the
N
′ gene confers hypersensitive resistance to some strains of tobacco mosaic virus (TMV) but not to the common strain. TMV sequences responsible for inducing local lesion formation in this host were identified by using cDNA clones to construct genomic recombinants between the common strain genome and a local-lesion-inducing mutant. To assay for sequences conferring the mutant phenotype,
in vitro
transcripts of recombinants were inoculated onto leaves of
N. sylvestris
and observed for the formation of either local lesions or a systemic infection. Sequences from the mutant that converted the hybrid genome to the mutant phenotype were located between nucleotides 5972 and 6206. Sequence analysis of this region revealed point mutations in the mutant at nucleotides 6157 (cytosine to uracil) and 6199 (adenine to guanine). The mutation at 6157 changes the capsid protein gene to specify phenylalanine rather than serine at position 148; nucleotide 6199 occurs in the 3′ nontranslated region. When each point mutation was individually substituted into the wild-type background, transcripts containing only the alteration at 6157 produced local lesions on
N. sylvestris
, whereas transcripts containing only the alteration at 6199 produced systemic mosaic symptoms. The frequency of mutation was examined by partially sequencing virion RNA from six additional independent local-lesion mutants. Five mutants had the same alteration at 6157 as the original mutant and none had the alteration at 6199. This work demonstrates that the capsid protein gene of TMV is multifunctional, both encoding the virion structural protein and mediating the outcome of infection in
N. sylvestris
.
Hepatitis B virus suppresses expression of human beta-interferon.
Authors: Twu J S , Lee C H , Lin P M , Schloemer R H
Published: May 2006

To determine whether hepatitis B virus (HBV) regulates the expression of the human beta-interferon gene, a series of recombinant bovine papilloma virus plasmids containing the human interferon gene and various fragments of the HBV genome were constructed and used to transform C127 cells, a murine fibroblast line. Analysis of the DNA from transformed C127 cells indicated that the interferon gene was intact and that the plasmids replicated as stable multicopy elements. The 1828-base-pair BamHI HBV DNA fragment containing the core antigen gene, but not the 2755-base-pair Bgl II HBV DNA fragment encoding both the surface antigen and the X antigen, suppressed the production of human beta-interferon. No effect by any of the recombinant plasmids on the synthesis of murine interferon was detected. The suppression of human beta-interferon by HBV occurs via a trans-acting factor. A frameshift mutation within the HBV core gene alleviates the inhibitory activity; thus we infer that the core protein is this factor or is crucially associated with this activity.