bluuebunny/crossref_metadata_2025 huggingface dataset:
Aluminum toxicity and forest decline
Authors: Godbold D. L. , Fritz E. , Hüttermann A.
Published: May 2006

The rate of root elongation in seedlings of
Picea abies
was drastically inhibited by exposure to AlCl
3
at 800 or 1200 μmol/dm
3
in nutrient solutions. A 35-day exposure to Al at 700 μmol/dm
3
reduced Mg and Ca in roots and needles of the seedlings. The Mg content of needles was reduced to levels considered to be critical for Mg deficiency. In investigations of
45
Ca uptake into roots, exposure to Al at 100-800 μmol/dm
3
reduced
45
Ca uptake by 77-92%, respectively. By using x-ray microanalysis, the distribution of Al, Mg, Ca, and K was found to be similar in roots of
Picea abies
seedlings grown in solution culture and in roots collected from declining spruce stands at Solling, F.R.G. In solution culture Al displaced Mg and Ca in the root cortex. A mechanism of Al toxicity for root growth and ion uptake is proposed, and its relevance to forest decline is discussed.
Nuclear fusion-defective phenocopies in Chlamydomonas reinhardtii: mating-type functions for meiosis can act through the cytoplasm.
Authors: Dutcher S K
Published: May 2006

Nuclear fusion in newly formed Chlamydomonas reinhardtii zygotes can be inhibited by drugs that affect microtubule stability, which include colchicine, amiprophosmethyl, oryzalin, and taxol. This inhibition can be monitored genetically by the production of haploid meiotic products from conjugations between haploid and diploid parents. Such zygotes would normally produce aneuploid progeny. Inhibition of nuclear fusion by colchicine requires treatment of gametic cells both before conjugation and after formation of the zygotes. These results suggest that nuclear fusion requires dynamic microtubules. Treated zygotes formed from a haploid-diploid mating can produce six spores, but only four spores germinate to form viable haploid colonies. No contribution from the nuclear genome of the haploid parent is recovered, whereas all loci from the diploid parent are recovered. The four viable products from the diploid parent of inhibited zygotes show normal segregation of loci located on linkage groups segregating according to Mendelian laws. Levels of meiotic recombination were tested for pairs of loci on linkage groups XVIII and XIX and found to be unchanged by inhibition of nuclear fusion. Thus, similar to Saccharomyces cerevisiae, C. reinhardtii mating-type functions necessary for nuclear fusion are not nuclear limited and can act through the cytoplasm. Inhibition of nuclear fusion can be used to analyze diploid Chlamydomonas that cannot enter meiosis. This technique permits direct analysis of dominant mutations, dominant suppressors and enhancers, and new alleles of identified loci that have been isolated in diploid strains.
Splice junction mutation in some Ashkenazi Jews with Tay-Sachs disease: evidence against a single defect within this ethnic group.
Authors: Myerowitz R
Published: May 2006

Tay-Sachs disease is an inherited disorder in which the alpha chain of the lysosomal enzyme beta-N-acetylhexosaminidase A bears the mutation. Ashkenazi Jews are found to be carriers for a severe type of Tay-Sachs disease, the classic form, 10 times more frequently than the general population. Ashkenazi Jewish patients with classic Tay-Sachs disease have appeared to be clinically and biochemically identical, and the usual assumption has been that they harbor the same alpha-chain mutation. In this study I have isolated the alpha-chain gene from an Ashkenazi Jewish patient, GM2968, with classic Tay-Sachs disease and compared its nucleotide sequences with that of the normal alpha-chain gene in the promoter region, exon and splice junction regions, and polyadenylylation signal area. Only one difference was observed between these sequences: at the 5' boundary of intron 12, a guanosine in the conserved splice junction dinucleotide sequence G-T had been altered to a cytidine. The alteration is presumed to be functionally significant and to result in aberrant mRNA splicing. Utilizing the polymerase chain reaction to amplify the region encompassing the mutation, I developed an assay to screen patients and heterozygote carriers for this mutation. Surprisingly, in each of two Ashkenazi patients, only one alpha-chain allele harbored the splice junction mutation. Only one parent of each of these patients was positive for the defect. Another Ashkenazi patient did not bear this mutation at all nor did either of the subject's parents. In addition, 30% of obligate heterozygotes tested carried the splice junction mutation, whereas 20 Ashkenazi Jews designated noncarriers by enzymatic assay were negative for this alteration. The data are consistent with the presence of more than one mutation underlying the classic form of Tay-Sachs disease in the Ashkenazi Jewish population.
Specific and stable labeling of antibodies with technetium-99m with a diamide dithiolate chelating agent.
Authors: Fritzberg A R , Abrams P G , Beaumier P L , Kasina S , Morgan A C , Rao T N , Reno J M , Sanderson J A , Srinivasan A , Wilbur D S
Published: May 2006

Technetium-99m labeling of antibodies has been suboptimal because of low affinity adventitious binding, nonspecific labeling, and loss of immunoreactivity. The diamide dithiolate ligand system (N2S2) forms highly stable, well-defined tetradentate complexes with Tc(V). Antibodies and their fragments have been labeled by conjugation of preformed 99mTc-4,5-bis(thioacetamido)pentanoate active ester to protein amine groups to give a chemically known 99mTc-N2S2 complex covalently linked to antibody. Evaluations of the 99mTc-N2S2-bound antibodies and their fragments have shown high stability and retained immunoreactivity.
Relationship between force and Ca2+ concentration in smooth muscle as revealed by measurements on single cells.
Authors: Yagi S , Becker P L , Fay F S
Published: May 2006

The role of Ca2+ in regulating smooth muscle contraction was investigated by measuring isometric force and [Ca2+] simultaneously in individual single smooth-muscle cells. [Ca2+] was measured with fura-2 and a high time-resolution dual-wavelength digital microfluorimeter, and force was measured with an ultrasensitive force transducer attached to a probe around which was tied one end of the cell. Both [Ca2+] and force increase after maximal electrical stimulus, with [Ca2+] increasing considerably before the first detectable increase in force. Force development exhibited maximal sensitivity to [Ca2+] between 150 and 500 nM Ca2+. This Ca2+ sensitivity can account for the fact that many physiological stimuli produce full contraction even though such stimuli only increase Ca2+ to 600-800 nM. When Ca2+ was induced to increase rapidly, the relation between [Ca2+] and force exhibited hysteresis. During the onset of contraction, force at a given [Ca2+] was lower than during the muscle's return to rest, thus suggesting the existence of a slow step(s) linking Ca2+ and force development in smooth muscle. The direction of this hysteresis reversed during contractions in which Ca2+ increased slowly, suggesting that the contractile process becomes desensitized to [Ca2+] with time. These relations between calcium and force in intact single smooth-muscle cells differ in many respects from the relation found previously in chemically permeabilized multicellular preparations of smooth muscle.
Potential positive and negative autoregulation of p60c-src by intermolecular autophosphorylation.
Authors: Cooper J A , MacAuley A
Published: May 2006

The product of the protooncogene c-src is a protein-tyrosine kinase, p60c-src, that is normally inhibited by phosphorylation at a tyrosine residue close to the C terminus (Tyr-527). If activated by dephosphorylation of Tyr-527, or by other means, p60c-src becomes phosphorylated at a tyrosine residue in the catalytic domain (Tyr-416). To test whether either or both of these tyrosines can be phosphorylated by p60c-src itself, we have created four mutations in c-src. One mutant product can receive but cannot donate phosphate, and other mutants are capable of catalysis but lack phosphorylation sites. The mutant genes were expressed singly or in combination in yeast. Analysis of the phosphorylation of mutant p60c-src in the yeast cells and in immunoprecipitates showed that p60c-src molecules can phosphorylate each other at Tyr-416 and -527. Prohibiting intramolecular phosphorylation had little effect on reaction rates and extents, suggesting that intermolecular phosphorylation predominates. If the same situation pertains in the milieu of the vertebrate fibroblast, phosphorylation of one p60c-src by another at Tyr-416 or -527 could permit positive or negative autoregulation.
Mutation of a site of tyrosine phosphorylation in the lymphocyte-specific tyrosine protein kinase, p56lck, reveals its oncogenic potential in fibroblasts.
Authors: Amrein K E , Sefton B M
Published: May 2006

p56lck, a cellular tyrosine protein kinase (EC 2.7.1.112) of the src family, is expressed in essentially all T cells and in some B cells. Expression in nonlymphoid cells is observed only rarely. We have found that mutation of a carboxyl-terminal phosphorylation site, tyrosine-505, reveals an oncogenic activity of this protein. Infection of fibroblasts with a retrovirus encoding wild-type p56lck is without consequence. In contrast, infection with a virus encoding the mutant protein leads to greatly increased phosphorylation of cellular proteins on tyrosine, morphological transformation, and anchorage-independent growth. This suggests that the tyrosine protein kinase activity and the oncogenic potential of p56lck are normally suppressed in vivo by phosphorylation of tyrosine-505. Since similar results were obtained previously with an analogous mutant of c-src, our results suggest that the protein kinase activity of all members of the src family of cytoplasmic tyrosine protein kinases will prove to be regulated by tyrosine phosphorylation at a conserved residue near the carboxyl terminus. Because p56lck is normally expressed only in lymphoid cells, it was possible that p56lck would be without effect in other tissues. The transformation of fibroblasts by mutant p56lck shows that this lymphoid protein can interact productively with nonlymphoid polypeptide substrates.
NIH-3T3 cells transformed by the EJ-ras oncogene exhibit reduced platelet-derived growth factor-mediated Ca2+ mobilization.
Authors: Benjamin C W , Connor J A , Tarpley W G , Gorman R R
Published: May 2006

NIH-3T3 cells transformed by the EJ-ras oncogene synthesize only 10-15% as much inositol 1,4,5-trisphosphate (InsP3) as control cells after stimulation with platelet-derived growth factor (PDGF). This is despite the fact that the basal (unstimulated) levels of InsP3 synthesized in control and EJ-ras-transformed cells are not significantly different. Using the fluorescent indicator fura-2 and digital-imaging techniques, we have visualized and quantified changes in intracellular Ca2+ concentrations in control and EJ-ras-transformed NIH-3T3 cells in response to PDGF. Within 3 min after exposure of control cells to PDGF, intracellular Ca2+ levels are increased 3- to 9-fold, paralleling the increase in InsP3. In contrast, the majority (greater than 90%) of the EJ-ras-transformed cells show no increase in Ca2+ levels after PDGF exposure and the few that did respond exhibited only a small transient increase. Pronounced differences in the intracellular localization of Ca2+ increases in control and the responding EJ-ras-transformed cells were also observed. Despite the inhibition of InsP3 synthesis and subsequent Ca2+ mobilization, the EJ-ras-transformed cells respond mitogenically to PDGF. These data do not support the hypothesis that the EJ-ras gene product (p21) stimulates a phosphatidylinositol 4,5-bisphosphate-specific phospholipase C in NIH-3T3 cells; instead they suggest that the EJ-ras p21 may uncouple the PDGF receptor from phospholipase C resulting in inhibition of PDGF-stimulated activity of phospholipase C, InsP3 synthesis, and Ca2+ mobilization.
A cDNA encoding tyrosinase-related protein maps to the brown locus in mouse.
Authors: Jackson I J
Published: May 2006

A mouse melanoma cDNA clone was isolated by virtue of its reactivity with two antisera raised against tyrosinase (EC 1.14.18.1) from two species, hamster and mouse. The cDNA (5A) cross-hybridizes with another, pMT4 [Shibahara, S., Tomita, V., Sakakura, T., Nager, C., Bhabatosh, C. & Muller, R. (1986) Nucleic Acids Res. 14, 2413-2427], previously thought to encode mouse tyrosinase. Two other cDNAs, one human and one mouse, have been reported recently [Kwon, B. S., Haq, A. K., Pomerantz, S. H. & Halaban, R. (1987) Proc. Natl. Acad. Sci. USA 84, 7473-7477; and Yamamoto, H., Takeuchi, S., Kudo, T., Makino, K., Nakata, A., Shinoda, T. & Takeuchi, T. (1987) Jpn. J. Genet. 62, 271-277] as candidates for tyrosinase, and they map at or very close to the mouse albino (c) locus. The proteins they encode are very similar to each other but are distinct from (although related to) the pMT4-encoded protein. Here I use recombinant inbred strains to localize pMT4 at or close to the mouse brown (b) locus. I suggest that the gene mapping to c is the authentic tyrosinase gene, whereas that mapping to b encodes a tyrosinase-related protein. All b mutations in laboratory strains are associated with the same diagnostic Taq I fragment, suggesting that all derive from the same original mutation. I discuss possible function(s) of the tyrosinase-related protein.
Site-specific binding of viral plus single-stranded RNA to replicase-containing open virus-like particles of yeast.
Authors: Esteban R , Fujimura T , Wickner R B
Published: May 2006

X double-stranded RNA is a deletion mutant of L-A double-stranded RNA and is encapsidated in viral particles by the L-A-encoded major coat protein. X double-stranded RNA has all the cis sites necessary to be transcribed, encapsidated, and replicated. We have cloned X double-stranded RNA and sequenced it. The complete X double-stranded RNA sequence deduced indicates that the first 25 bases of the X plus-strand 5' end originated from the 5' end of the L-A plus strand and that most, if not all, of the rest comes from the 3' end of the L-A plus strand. The X plus strand made by X double-stranded RNA-containing virus-like particles binds specifically to empty open virus-like particles and is converted by these particles to X double-stranded RNA. RNA transcripts of the X complementary DNA clones and deletion derivatives thereof were made in vitro by T7 and T3 RNA polymerases and tested for specific binding to the virus-like particles. The results suggest that the binding is due to the sequence UUUGGCCAGG, 370 bases upstream from the X plus-strand 3' end. This sequence is also present in the M1 plus strand 140 bases from its 3' end.
Human immunodeficiency virus type 1 neutralization epitope with conserved architecture elicits early type-specific antibodies in experimentally infected chimpanzees.
Authors: Goudsmit J , Debouck C , Meloen R H , Smit L , Bakker M , Asher D M , Wolff A V , Gibbs C J , Gajdusek D C
Published: May 2006

Chimpanzees are susceptible to infection by divergent strains of human immunodeficiency virus type 1 (HIV-1), none of which cause clinical or immunological abnormalities. Chimpanzees were inoculated with one of four strains of HIV-1: human T-lymphotropic virus (HTLV) type IIIB, lymphadenopathy virus (LAV) type 1, HTLV type IIIRF, or an isolate from the brain of a patient with acquired immunodeficiency syndrome. Within 6 months after inoculation with the closely related strains HTLV-IIIB or LAV-1, six chimpanzees developed serum antibodies to the C-terminal half (amino acids 288-467) of the HTLV-IIIB external envelope glycoprotein gp120. Sera from five of those chimpanzees had HTLV-IIIB cell-fusion-inhibiting antibody titers greater than or equal to 20 at that time, indicating that they neutralized the infecting strain of HIV-1 in vitro. No antibodies to the carboxyl terminus of HTLV-IIIB gp120 were observed in sera of chimpanzees inoculated with HTLV-IIIRF or with the brain-tissue strain, and those sera did not neutralize HTLV-IIIB. A rabbit immunized with the C-terminal portion of gp120 acquired neutralizing antibodies that bound to four domains of the HTLV-IIIB external envelope as analyzed by reactivity to 536 overlapping nonapeptides of gp120. One of these domains in the variable region V3, with the amino acid sequence IRIQRGPGRAFVTIG (amino acids 307-321), bound to all chimpanzee sera that neutralized HTLV-IIIB but not to the serum of the HTLV-IIIRF-inoculated chimpanzee that did not neutralize HTLV-IIIB. The HTLV-IIIRF sequence at the same location, ITKGPGRVIYA, was recognized by the serum of the HTLV-IIIRF-inoculated chimpanzee but not by any sera of the HTLV-IIIB-inoculated or LAV-1-inoculated chimpanzees. The HTLV-IIIB residues RIQR and AFV and the HTLV-IIIRF residues lysine and VIYA, flanking a highly conserved beta-turn (GPGR), appear to be critical for antibody binding and subsequent type-specific virus neutralization. This neutralization epitope, putatively consisting of a loop between two cysteine residues (amino acids 296 and 331) connected by a disulfide bond, is immunodominant in HIV-1-infected chimpanzees and induces antibodies restricted to the homologous viral strain.
Tunichlorin: a nickel chlorin isolated from the Caribbean tunicate Trididemnum solidum.
Authors: Bible K C , Buytendorp M , Zierath P D , Rinehart K L
Published: May 2006

Tunichlorin, a blue-green pigment isolated from the Caribbean tunicate Trididemnum solidum, has been identified as nickel(II) 2-devinyl-2-hydroxymethylpyropheophorbide a by chemical and spectroscopic methods, with confirmation by partial synthesis of dimethyl tunichlorin from chlorophyll a. Nickel chlorins have been reported from geological sources but not from living organisms. Its occurrence in a living system suggests a metabolic role for tunichlorin and may clarify the selective accumulation of nickel by marine tunicates. Because Trididemnum tunicates are associated with algal symbionts, tunichlorin may arise directly from the tunicate, from symbiotic algae, or from tunicate modification of an algal chlorin.
A Holliday recombination intermediate is twofold symmetric.
Authors: Churchill M E , Tullius T D , Kallenbach N R , Seeman N C
Published: May 2006

Four-arm Holliday structures are ephemeral intermediates in genetic recombination. We have used an oligodeoxynucleotide system to form immobile DNA junctions, which are stable analogs of Holliday structures. We have probed the equilibrium structure of a junction by means of hydroxyl radicals generated by the reaction of iron(II)EDTA with hydrogen peroxide. The hydroxyl radical cleavage pattern shows twofold symmetry throughout the molecule. Strong protection from hydroxyl radical attack is evident on two strands near the branch site, and weaker protection may be seen four or five residues 3' to the branch site on the other two strands. No other position appears significantly distinct from double-helical DNA controls. From these data, we conclude that the Holliday junction is a twofold symmetric complex whose four arms form two stacking domains.
Sources and evolution of human Alu repeated sequences.
Authors: Britten R J , Baron W F , Stout D B , Davidson E H
Published: May 2006

Alu repeated sequences arising in DNA of the human lineage during about the last 30 million years are closely similar to a modern consensus. Alu repeats arising at earlier times share correlated blocks of differences from the current consensus at diagnostic positions in the sequence. Using these 26 positions, we can recognize four subfamilies and the older ones are each successively closer to the 7SL sequence. It appears that there has existed a series of conserved genes that are the primary sources of the Alu repeat family, presumably through retroposition. These genes have probably replaced each other in overlapping relays during the evolution of primates.
Cytoplasmic domain affects membrane expression and function of an Ia molecule.
Authors: Griffith I J , Ghogawala Z , Nabavi N , Golan D E , Myer A , McKean D J , Glimcher L H
Published: May 2006

The association of foreign antigen with Ia molecules on the surface of antigen-presenting cells is necessary for the interaction with the clonally distributed antigen receptor on T cells and is therefore critical in the initiation and regulation of immune responses. Ia polypeptides (alpha and beta) are composed of two extracellular domains, a transmembrane domain and a cytoplasmic domain. Although exon-shuffling experiments have demonstrated that antigen associates with the NH2-terminal alpha 1 and beta 1 domains, the roles that the other domains play in Ia function are still poorly understood. The B-hybridoma cell line 2B1 was selected in a series of positive and negative immunoselection steps for a mutation in the Ek alpha polypeptide. It was found to fortuitously contain a mutation in the Ak alpha polypeptide as well. Sequence analysis of the Ak alpha gene showed that a single base transition (C----T) resulted in a stop codon at amino acid residue 222. This caused the loss of 12 amino acids from the cytoplasmic domain of the mature polypeptide. This mutation results in a decreased level of Ak alpha polypeptide expression on the cell surface (50% of wild-type levels), an increased half-life of Ak alpha polypeptide in the cell, and a specific limited defect in antigen presentation.
Differential distribution of D and L amino acids between the 2' and 3' positions of the AMP residue at the 3' terminus of transfer ribonucleic acid.
Authors: Lacey J C , Hawkins A F , Thomas R D , Watkins C L
Published: May 2006

Amino acids esterified to the ribose group of 5'-adenylic acid (AMP) constantly migrate between the 2' and 3' positions of the ribose at a rate of several times per second, which is slower than the rate of peptide-bond synthesis (15-20 per sec). Because the contemporary protein-synthesizing system only incorporates amino acids into protein when they are at the 3' position of the AMP at the terminus of tRNA, the value of the equilibrium constant relative to the 2' and 3' positions is of considerable interest. Differences between D and L isomers in this regard might be especially revealing. We have used N-acetylaminoacyl esters of AMP as models for the 3' terminus of tRNA and find that glycine and the L amino acids consistently distribute predominantly to the 3' position (approximately equal to 67% 3', approximately equal to 33% 2'), but D amino acids distribute to that position generally to a lesser extent and in a manner inversely related to the hydrophobicity of the amino acid side chain. This consistency of the L amino acid preference for the 3' position, combined with the inconsistency of the D amino acid preference, may be one reason for the origin of our contemporary protein-synthesizing system, which forms the peptide bond preferentially with L amino acids and only when they are in the 3' position of the ribose moiety of the AMP residue at the 3' terminus of every tRNA.
Cytokinin gene fused with a strong promoter enhances shoot organogenesis and zeatin levels in transformed plant cells
Authors: Smigocki A. C. , Owens L. D.
Published: May 2006

The isopentenyltransferase (
ipt
) gene associated with cytokinin biosynthesis in plants was cloned from a tumor-inducing plasmid carried by
Agrobacterium tumefaciens
and placed under the control of promoters of differing activities, the cauliflower mosaic virus 35S promoter and the nopaline synthase promoter. These promoter-gene constructs were introduced into wounded
Nicotiana
stems, leaf pieces, and cucumber seedlings by
A. tumefaciens
infection. Shoots were observed in the infection site on all responding genotypes of
Nicotiana
plants infected with the 35S promoter construct (35S—
ipt
), whereas only 41% responded similarly to infection with the unmodified gene. Furthermore, shoots were observed 19 days after infection with the 35S—
ipt
gene but not until 28 to 45 days with the unaltered
ipt
gene. Shoots were more numerous (>40) on galls incited by 35S—
ipt
and were up to 6 times taller than shoots induced by the native gene. On
Cucumis
(cucumber), shoots were observed only on galls incited by the 35S—
ipt
construct. These galls were on the average 7.5 times larger than those incited by the nopaline synthase promoter construct (NOS—
ipt
) or the unmodified
ipt
gene. Zeatin and zeatinriboside concentrations averaged 23 times greater in the 35S—
ipt
transformed shoots than in ones transformed with the native
ipt
gene. These results suggest that a more active promoter on the
ipt
gene can enhance or change the morphogenic potential of transformed plant cells by increasing their endogenous cytokinin levels.
Path integration in desert ants,
Cataglyphis fortis
Authors: Müller Martin , Wehner Rüdiger
Published: May 2006

Foraging desert ants,
Cataglyphis fortis
, continually keep track of their own posotions relative to home— i.e., integrate their tortuous outbound routes and return home along straight (inbound) routes. By experimentally manipulating the ants' outbound trajectories we show that the ants solve this path integration problem not by performing a true vector summation (as a human navigator does) but by employing a computationally simple approximation. This approximation is characterized by small, but systematic, navigational errors that helped us elucidate the ant's way of computing its mean home vector.
Lateral geniculate lesions block circadian phase-shift responses to a benzodiazepine.
Authors: Johnson R F , Smale L , Moore R Y , Morin L P
Published: May 2006

Several pharmacological treatments, including application of an excitatory neurotoxin to the lateral geniculate nucleus (LGN) and systemic administration of triazolam, a clinically effective benzodiazepine, can elicit large phase shifts in a circadian rhythm according to the time of administration. The hypothesis that the LGN might mediate the effect of triazolam on circadian clock function was tested. Bilateral lesions of the LGN, which destroyed the connection from the intergeniculate leaflet to the suprachiasmatic nucleus, blocked phase-shift responses to triazolam. The requirement of an intact LGN for triazolam to shift circadian phase suggests that the LGN may be a site through which stimuli gain access to the circadian clock to modulate rhythm phase and entrainment.
Inositol phospholipid-specific phospholipase C: complete cDNA and protein sequences and sequence homology to tyrosine kinase-related oncogene products.
Authors: Suh P G , Ryu S H , Moon K H , Suh H W , Rhee S G
Published: May 2006

Antibodies against an inositol phospholipid-specific phospholipase C purified from bovine brain were used to screen rat brain lambda gt11 expression cDNA libraries. Complete sequences of three cDNA inserts yielded a cumulative sequence of 5106 base pairs. The deduced protein had 1289 amino acids with a calculated molecular weight of 148,431. The determination of an open reading frame was aided by the amino acid sequences of 21 tryptic peptides isolated from bovine brain phospholipase C. Only 9 residues of a total of 140 amino acid residues determined for the bovine enzyme were different from those deduced from the rat cDNA. Two regions of phospholipase C (amino acid residues 555-598 and 668-705) exhibited significant amino acid similarities to the products of various tyrosine kinase-related oncogenes (yes, src, fgr, abl, fps, fes, and tck). The homologous domain was located in the region that is not essential for the protein-tyrosine kinase activity but is likely to be involved in an interaction with cellular components that modulate kinase function. Therefore, this unexpected similarity raises the possibility that the 148-kDa phospholipase C and cytoplasmic tyrosine kinases are modulated by common cellular component(s).
Self-incompatibility genes of
Brassica oleracea
: Expression, isolation, and structure
Authors: Nasrallah June B. , Yu Su-May , Nasrallah Mikhail E.
Published: May 2006

We demonstrate by
in situ
hybridization the cell-type-specific expression of transcripts encoded by the self-incompatibility (
S
) locus of
Brassica oleracea
. These transcripts are not detected early in stigma development, their expression is switched on in the papillar cells of the stigma surface, and their levels increase in these cells in correlation with the acquisition by the stigma of the self-incompatibility response. By using a probe derived from the untranslated sequences at the 3′ end of
S
cDNA, an
S
-gene copy expressed in the papillar cells has been isolated from among the multiple
S
-related copies that occur in the
Brassica
genome. Structural analysis of this gene shows that it lacks introns. In light of the strict spatial and temporal regulation of
S
-gene expression in precisely the cells that constitute the barrier to self-pollination, the self-incompatibility response may be viewed as a cell-cell interaction between one pollen grain and one papillar cell.
Molecular basis of human growth hormone gene deletions.
Authors: Vnencak-Jones C L , Phillips J A , Chen E Y , Seeburg P H
Published: May 2006

Crossover sites resulting from unequal recombination within the human growth hormone (GH) gene cluster that cause GH1 gene deletions and isolated GH deficiency type 1A were localized in nine patients. In eight unrelated subjects homozygous for 6.7-kilobase (kb) deletions, the breakpoints are within two blocks of highly homologous DNA sequences that lie 5' and 3' to the GH1 gene. In seven of these eight cases, the breakpoints map within a 1250-base-pair (bp) region composed of 300-bp Alu sequences of 86% homology and flanking non-Alu sequences that are 600 and 300 bp in length and are of 96% and 88% homology, respectively. In the eighth patient, the breakpoints are 5' to these Alu repeats and are most likely within a 700-bp region of 96% homologous DNA sequences. In the ninth patient homozygous for a 7.6-kb deletion, the breakpoints are contained within a 29-bp perfect repeat lying 5' to GH1 and the human chorionic somatomammotropin pseudogene (CSHP1). Together, these results indicate that the presence of highly homologous DNA sequences flanking GH1 predispose to recurrent unequal recombinational events presumably through chromosomal misalignment.
Isolation of cDNAs of scrapie-modulated RNAs by subtractive hybridization of a cDNA library.
Authors: Duguid J R , Rohwer R G , Seed B
Published: May 2006

We have developed a subtractive cloning procedure based on the hybridization of single-stranded cDNA libraries constructed in pi H3M, a vector containing the phage M13 origin of replication. We have used this strategy to isolate three transcripts whose abundance is increased in scrapie-infected brain. DNA sequence analysis showed that they represent glial fibrillary acidic protein, metallothionein II, and the B chain of alpha-crystallin; the latter two may represent a response to stress.
Voltage dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in peeled skeletal muscle fibers.
Authors: Donaldson S K , Goldberg N D , Walseth T F , Huetteman D A
Published: May 2006

Excitation-contraction coupling in skeletal muscle is known to be under absolute control of plasmalemma voltage, but the steps from transverse (T)-tubule depolarization to Ca2+ release from the sarcoplasmic reticulum have not been elucidated. The effect of changing T-tubule membrane potential on inositol 1,4,5-trisphosphate (InsP3) stimulation of Ca2+ release from the sarcoplasmic reticulum was studied to explore a possible role for InsP3 as a chemical signal in excitation-contraction coupling. InsP3 was microinjected into peeled rabbit skeletal muscle fibers at a pipette concentration of 0.5 microM; Ca2+ release from the sarcoplasmic reticulum was monitored as an isometric tension transient. The response to 0.5 microM InsP3 was significantly larger when T-tubules were in a depolarized state than when they were in a polarized state, and this difference in response was independent of the ionic composition of the bathing solutions or the method for depolarizing the T-tubules. Thus, T-tubule depolarization may sensitize the sarcoplasmic reticulum to a preexisting low concentration of InsP3 and greatly reduce the need for InsP3 production. Plasmalemma voltage control of the stimulatory effects of InsP3 may have relevance for mechanisms in excitable nonmuscle cells.
Calcium oscillations in single adrenal glomerulosa cells stimulated by angiotensin II.
Authors: Quinn S J , Williams G H , Tillotson D L
Published: May 2006

The cytosolic calcium (Ca2+i) response to angiotensin II (Ang II) was examined in single rat zona glomerulosa cells by monitoring fura-2 fluorescence with microspectrofluorimetry. Ang II concentrations ranged from 5 X 10(-12) to 5 X 10(-8) M. The mean peak Ca2+i increase was similar at all Ang II concentrations (205 +/- 11 nM), with a significant difference (P less than 0.05) found only between 5 X 10(-12) M (151 +/- 16 nM) and 5 X 10(-9) M (236 +/- 24 nM). Striking differences over the range of Ang II concentrations were found in the Ca2+i response kinetics. A dose-dependent delay of the onset of the Ca2+i response was observed ranging from 2.6 +/- 0.3 sec at 5 X 10(-8) M to 181 +/- 27 sec at 5 X 10(-12) M Ang II. After the delay, cells typically responded with an abrupt increase in Ca2+i, complete within 15 sec. At low Ang II concentrations (5 X 10(-11) and 5 X 10(-12) M), a complex response was often observed consisting of Ca2+i oscillations. Higher Ang II concentrations gave some evidence of Ca2+i oscillation, especially at 5 X 10(-10) M where oscillations appeared fused. Above 5 X 10(-10) M Ang II, the initial Ca2+i increase decayed to an apparent steady-state value 38-40% of the peak response within 5 min; 5 X 10(-10) M Ang II produced a smaller decline to 63% of the initial Ca2+i increase. In contrast to cell population studies, assessment of individual glomerulosa cells demonstrates (i) a dose-dependent delay prior to a rapid increase in Ca2+i; (ii) a similar peak increase at most Ang II concentrations; (iii) greater sensitivity of the Ca2+i response; and (iv) a complex oscillating Ca2+i response in the physiological range of Ang II.
p21ras-induced responsiveness of phosphatidylinositol turnover to bradykinin is a receptor number effect.
Authors: Downward J , de Gunzburg J , Riehl R , Weinberg R A
Published: May 2006

Proteins encoded by ras genes have recently been reported to couple certain growth factor receptors to phospholipase C, the enzyme catalyzing phosphatidylinositol breakdown. To investigate this hypothesis, the normal and the transforming Ha-, Ki-, and N-ras genes were each transfected into Rat-1 fibroblasts under the control of strong promoters. Several cell lines, both normal and transformed, were selected that expressed high levels of p21ras. Phosphatidylinositol turnover was measured in these cells in response to a wide variety of peptide factors; bradykinin was found to have a greatly enhanced effect on the p21ras overexpressors relative to the parental and control cells. Bradykinin receptor numbers were measured in these lines and found to be up to 40-fold higher in the p21ras overexpressors than in the parental cells. This was found to be the case for both normal and transforming forms of all three varieties of ras genes. Receptor number correlated well with the bradykinin-dependent phosphatidylinositol turnover response in all cases. These data indicate that the effects of p21ras on cellular responses to the peptide hormone bradykinin are due to changes in receptor number rather than to direct coupling by p21ras between the receptor and phospholipase C.
Characterization of the gene for the a subunit of human factor XIII (plasma transglutaminase), a blood coagulation factor.
Authors: Ichinose A , Davie E W
Published: May 2006

Factor XIII (plasma transglutaminase, fibrin stabilizing factor) is a glycoprotein that circulates in blood as a tetramer (a2b2) consisting of two a and two b subunits. The primary structures of the a and b subunits of human factor XIII have been reported by a combination of cDNA cloning and amino acid sequence analysis. To establish the gene structure of the a subunit for factor XIII, several human genomic libraries were screened by using the cDNA encoding the a subunit as a probe. Among approximately equal to 5 x 10(7) recombinant phage, 121 have been shown to contain an insert encoding a portion of the a subunit. Twenty-five unique clones were then characterized by restriction mapping, Southern blotting, and DNA sequencing. Overlapping clones encoding the a subunit of factor XIII span greater than 160 kilobases. The gene was found to contain 15 exons separated by 14 introns. All the sequences of the introns at the intron-exon boundaries were GT-AG, which are the same as those found in other eukaryotic genes. DNA sequence analysis revealed that the activation peptide released by thrombin, the active site cysteine region, the two putative calcium-binding regions, and the thrombin cleavage site leading to inactivation are encoded by separate exons. This suggests that the introns may separate the a subunit into functional and structural domains. A comparison of the amino acid sequence deduced from the genomic DNA sequence with those deduced from cDNA or determined by amino acid sequence analysis of the plasma and placental proteins revealed apparent amino acid polymorphisms in six positions of the polypeptide chain of the a subunit.
Activation of the polyomavirus enhancer by a murine activator protein 1 (AP1) homolog and two contiguous proteins.
Authors: Martin M E , Piette J , Yaniv M , Tang W J , Folk W R
Published: May 2006

The polyomavirus enhancer is composed of multiple DNA sequence elements serving as binding sites for proteins present in mouse nuclear extracts that activate transcription and DNA replication. We have identified three such proteins and their binding sites and correlate them with enhancer function. Mutation of nucleotide (nt) 5140 in the enhancer alters the binding site (TGACTAA, nt 5139-5145) for polyomavirus enhancer A binding protein 1 (PEA1), a murine homolog of the human transcription factor activator protein 1 (AP1). This mutation simultaneously reduces polyomavirus transcription and DNA replication. Reversion of this mutation simultaneously restores binding of PEA1 and both DNA replication and transcription. Binding of a second protein, PEA2, adjacent to the PEA1 site at nt 5147-5155 is enhanced by PEA1 binding, suggesting that these proteins interact. A third protein, PEA3, binds to the sequence AGGAAG (nt 5133-5138) adjacent to the PEA1 binding site; integrity of this late-proximal PEA3 binding site or an additional early-proximal site (nt 5228-5233) is important for enhancer function. We correlate binding of PEA1 and PEA2 with the induction of a DNase I-hypersensitive site in polyomavirus minichromosomes isolated from mouse fibroblasts.
Bovine papilloma virus-transformed cells contain multiple E2 proteins.
Authors: Hubbert N L , Schiller J T , Lowy D R , Androphy E J
Published: May 2006

Genetic evidence suggests that the bovine papilloma virus type 1 (BPV) E2 open reading frame may encode at least two gene products involved in the regulation of viral gene expression. One, which is probably the full-length product, trans-activates transcription via an enhancer in the viral regulatory region. A second, containing sequences from the 3' end of the open reading frame, inhibits the trans-activating activity of the first product. We now report the identification and initial characterization of three E2-encoded proteins, with mobilities corresponding to 48, 31, and 28 kDa in cells transformed by the wild-type BPV. Pulse-chase experiments indicated that the 48-kDa protein had the longest half-life (40 min), but there was no indication that one species was the precursor of another. The 48-kDa species corresponds to the full-length trans-activating protein. The two smaller species contain only carboxyl-terminal determinants, and either or both could represent inhibitory E2 proteins. Subcellular fractionation localized all three E2 proteins to the nucleus. Consistent with the low rate of viral transcription in BPV-transformed cells, the 31-kDa presumptive repressor species was more abundant than the 48-kDa species.
Cleavage furrow isolated from newt eggs: contraction, organization of the actin filaments, and protein components of the furrow.
Authors: Mabuchi I , Tsukita S , Tsukita S , Sawai T
Published: May 2006

The cleavage-furrow region was isolated surgically from newt eggs at the early stage of the first cleavage. The isolated furrow contracted in the presence of ATP at a Ca2+ concentration of 10 or 100 nM, although the speed was less than that of the furrow in vivo. Cytochalasin B, cytochalasin D, phalloidin, p-chloromercuribenzoate, and N-ethyl-maleimide interfered with the contraction, but colchicine did not. The furrow contained bundles of actin filaments of opposite polarities oriented parallel to the long axis of the furrow; these bundles may be the main component of the contractile arc. From electron microscopic observation of thin sections of the furrow, it was suggested that the actin bundles of the contractile arc were organized from preexisting cortical filaments that were connected to the plasma membrane by granular materials at their barbed ends. Contractile-arc actin filaments were revealed to be crosslinked by thin strands by the rapid freezing/deep etching-replication technique. Two-dimensional polyacrylamide gel electrophoresis showed that several proteins found in the furrow cortex are absent from the cortical layer before the cleavage furrow is formed.
cAMP antagonizes interleukin 2-promoted T-cell cycle progression at a discrete point in early G1.
Authors: Johnson K W , Davis B H , Smith K A
Published: May 2006

T lymphocytes are stimulated to proliferate in an autocrine/paracrine manner by the lymphokine interleukin 2 (IL-2). In seeking further insight into the mechanisms by which IL-2 induces progression of T cells through the G1 phase of the cell cycle, studies were performed with agents that increase cellular adenosine 3',5'-cyclic monophosphate (cAMP), a well-known inhibitor of lymphocyte growth. The addition of dibutyryl-cAMP, cholera toxin, forskolin, or 3-isobutyl-1-methylxanthine to an IL-2-dependent murine T-cell line evoked a dose-related suppression of S-phase transition without affecting cellular viability. Moreover, elevation of cAMP levels led to an accumulation of uniformly small cells, suggesting an arrest in early G1. Consistent with these findings, dibutyryl-cAMP inhibited the incorporation of both [3H]-uridine and [3H]thymidine by IL-2-stimulated, synchronized normal human T cells. Furthermore, maximal inhibition occurred during early G1, as indicated by experiments where the addition of dibutyryl-cAMP was delayed with respect to IL-2 stimulation. Quantitative flow cytometric analysis of RNA and DNA content of IL-2-stimulated cells affirmed that increased cAMP inhibits RNA accumulation and S-phase transition. In addition, exposure of IL-2-dependent, asynchronously proliferating normal human T cells to dibutyryl-cAMP resulted in uniform growth arrest in early G1, the point at which cycling T cells accumulate when they are deprived of IL-2. These results indicate that increased cAMP inhibits G1 progression stimulated by IL-2 and provide a rationale for the use of cAMP analogues as pharmacologic probes for the dissection of molecular events occurring during IL-2 signaling and T-cell G1 transit. They also suggest the possibility of therapeutic immunosuppression by a combination of agents that act at different stages of the T-cell cycle.
Autocrine secretion of tumor necrosis factor under the influence of interferon-gamma amplifies HLA-DR gene induction in human monocytes.
Authors: Arenzana-Seisdedos F , Mogensen S C , Vuillier F , Fiers W , Virelizier J L
Published: May 2006

Recombinant interferon-gamma (IFN-gamma) induced HLA-DR gene expression in both U937 and THP-1 human monocytic cell lines, although the former was only very weakly inducible. Combination of recombinant tumor necrosis factor (TNF) and IFN-gamma resulted in a synergistic enhancement of DR mRNA and protein induction in both cell lines. TNF alone increased the constitutive expression of the DR gene in THP-1 cells. In the HLA class II-negative U937 cells, TNF used alone was not able to induce DR gene expression. Such a negative result was not due to a lack of TNF receptor expression in U937 cells, since TNF clearly induced HLA class I and TNF gene expression in this cell line. THP-1, but not U937, cells secreted TNF under the influence of IFN-gamma. Neutralization of TNF by a specific antibody decreased IFN-gamma-induced DR antigen expression in THP-1 cultures. These observations indicate that TNF is not able to directly induce DR gene expression, but rather amplifies ongoing expression of this gene, whether constitutive or induced by IFN-gamma. In the two cell lines tested, the level of DR inducibility under the influence of IFN-gamma used alone depended on a different inducibility of TNF secretion by IFN-gamma. Altogether, our observations indicate that TNF, whether exogenous or endogenously produced under the influence of IFN-gamma, amplifies DR gene expression in monocytes, a phenomenon that may provide to such antigen-presenting cells a selective sensitivity to the DR-inducing effects of IFN-gamma.
Interleukin 4 induces membrane Thy-1 expression on normal murine B cells.
Authors: Snapper C M , Hornbeck P V , Atasoy U , Pereira G M , Paul W E
Published: May 2006

Thy-1, a cell-surface glycoprotein of undetermined function, is expressed in relatively large amounts on mouse thymocytes, peripheral T cells, and neurons. It is widely used as a marker to distinguish peripheral T cells from B cells in mice. We show here that, in five distinct mouse strains, recombinant interleukin 4 (IL-4/B-cell stimulatory factor 1) strikingly induces membrane expression of Thy-1 on the vast majority of lipopolysaccharide (LPS)-stimulated normal murine B cells. Thy-1+ B cells are precursors for immunoglobulin-secreting cells. RNA blot analysis indicates that B cells express a Thy-1 mRNA of 1.8 kilobases, the same size as that found in T cells. Cell mixing experiments show that only cells derived from Thy-1.2+ donors express Thy-1.2, indicating that B cells expressing Thy-1 have not passively absorbed the glycoprotein from another cell source. Recombinant interferon-gamma inhibits Thy-1 induction by B cells stimulated with LPS and IL-4. Thy-1 is also induced on B cells that have been stimulated as a result of the specific activation of an IL-4-producing T-helper clone. Anti-IL-4 monoclonal antibody inhibits the induction of B-cell Thy-1 in this T-cell-B-cell interaction.
LR16, a compound with potent effects on the oxygen affinity of hemoglobin, on blood cholesterol, and on low density lipoprotein.
Authors: Lalezari I , Rahbar S , Lalezari P , Fermi G , Perutz M F
Published: May 2006

2-[4-(3,4-Dichlorophenylureido)phenoxy]-2-methylpropionic acid, LR16, combines with two symmetrically related sites in the central cavity of deoxyhemoglobin, 20 A away from the binding site of 2,3-bisphosphoglycerate, and acts as an allosteric effector synergistic with 2,3-bisphosphoglycerate. LR16 (1 mM) raises P50, the partial pressure of oxygen needed to achieve half-saturation with oxygen of a hemolysate of human hemoglobin, about 50 times more strongly than 1 mM 2,3-bisphosphoglycerate. Oral administration of LR16 (at small doses that produced no ill effects) to rats that were fed a diet rich in cholesterol caused substantial reductions of total serum cholesterol and low density lipoprotein-cholesterol, while high density lipoprotein-cholesterol remained unchanged.