bluuebunny/crossref_metadata_2025 huggingface dataset:
Trans regulation of the phosphoenolpyruvate carboxykinase (GTP) gene, identified by deletions in chromosome 7 of the mouse.
Authors: Loose D S , Shaw P A , Krauter K S , Robinson C , Englard S , Hanson R W , Gluecksohn-Waelsch S
Published: May 2006

Livers from newborn mice homozygous for either one of the lethal deletions c14CoS or c3H in chromosome 7 have drastically reduced levels of cytosolic phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] activity when compared with normal littermates. The structural gene for the enzyme maps on chromosome 2 and appears intact and not grossly rearranged in deletion homozygotes. These mice also have negligible levels of hepatic mRNA encoding this enzyme. Studies of the transcription rate of the gene showed that it was reduced to 25-50% of normal in hepatic nuclei obtained from mice homozygous for either deletion. We suggest that, in addition to the reduction in the level of transcription, the deletions in chromosome 7 may also cause alterations in messenger stability, processing, or transport from the nucleus.
Genetic engineering of an H-2Dd/Q10b chimeric histocompatibility antigen: purification of soluble protein from transformant cell supernatants.
Authors: Margulies D H , Ramsey A L , Boyd L F , McCluskey J
Published: May 2006

We have constructed a recombinant class I gene in which 5' sequences of H-2Dd are linked to the 3' half of a Qa subregion gene, Q10b. This hybrid gene would be expected to direct the synthesis of a protein containing the N and C1 domains of H-2Dd covalently linked to the C2 domain of the secreted, nonpolymorphic, Q10b antigen. Following DNA-mediated gene transfer into mouse L cells, transformants were analyzed by radiolabeling and immunoprecipitation. These cells secreted a molecule reactive with anti-H-2Dd monoclonal antibodies that identify epitopes on the N and C1 domains as well as with an anti-Q10 carboxyl-terminal peptide antiserum. The H-2Dd-derived antigen is associated with beta 2-microglobulin and is readily purified in milligram amounts from culture supernatants by immunoaffinity chromatography.
Mechanism of cytotoxicity of human large granular lymphocytes: relationship of the cytotoxic lymphocyte protein to the ninth component (C9) of human complement.
Authors: Zalman L S , Brothers M A , Chiu F J , Müller-Eberhard H J
Published: May 2006

A Mr 70,000 protein was isolated from cytotoxic human large granular lymphocytes and shown to have cytotoxic activity. The protein was demonstrated to be immunochemically related to the ninth component (C9) of complement and was therefore designated C9-related protein (C9RP). This finding suggests that C9RP and C9 share homology in primary structure and have a common evolutionary ancestry. C9RP was isolated, by affinity chromatography employing anti-human C9-Sepharose, from either purified cytoplasmic granules or whole-cell lysates of cultured human large granular lymphocytes. The cells were isolated from healthy blood donors and maintained in interleukin-2-dependent cultures. The immunochemical crossreactivity of C9 with C9RP was 3-4%, using a murine anti-C9RP antiserum. Certain murine monoclonal antibodies to C9RP and to C9 inhibited killing of K562 cells by human large granular lymphocytes. Killed target cells, identified by propidium iodide staining and isolated by fluorescence-activated cell-sorting, exhibited clusters of circular membrane lesions that resembled poly(C9) in appearance. Polymerization of isolated C9RP in the presence of Ca2+ resulted in the formation of two different circular structures, one having an inner diameter of approximately equal to 60 A, and the other, of 125 A. Polymerized C9RP could be incorporated into liposomes and, as such, gave rise to channels of two different sizes. The smaller channel had a functional diameter of 50-90 A, and the bigger channel, a diameter greater than 102 A.
Deficiency of cyclic AMP-dependent protein kinases in human psoriasis.
Authors: Brion D E , Raynaud F , Plet A , Laurent P , Leduc B , Anderson W
Published: May 2006

To determine possible differences in the cyclic AMP-dependent protein kinases of normal and psoriatic human fibroblasts, the levels of the regulatory subunits (RI and RII, respectively) of protein kinase I and protein kinase II were quantitated by photoaffinity labeling with 8-azido[32P]cAMP. The level of RII was significantly decreased, or was undetectable, in cytosol prepared from fibroblasts from five psoriatic subjects when compared to RII levels found with normal human fibroblasts. The level of cytosolic RI was decreased in fibroblasts from four psoriatic patients and was within the normal range for one diseased patient when compared to RI levels in normal human fibroblasts. The elution profile from a DEAE-cellulose column of protein kinase activity in the soluble fraction from two psoriatic patients also showed a decrease in type I kinase activity and the complete absence of type II kinase activity. Other results indicate that the level of RI in erythrocyte membranes from psoriatic subjects is significantly decreased when compared to that of erythrocyte membranes from eight normal subjects. A significant correlation (P less than 0.001) was observed between the severity of the cutaneous manifestation of the disease and the level of RI in psoriatic erythrocyte membranes. The changes noted in the levels of RI and RII in cell types other than those thought to be specifically involved in the proliferative epidermis disorder of the disease suggest a general protein kinase deficiency.
Unregulated proliferation of primitive chronic myeloid leukemia progenitors in the presence of normal marrow adherent cells.
Authors: Eaves A C , Cashman J D , Gaboury L A , Kalousek D K , Eaves C J
Published: May 2006

Previous studies have shown that Philadelphia (Ph1) chromosome-positive chronic myeloid leukemia (CML) results from the abnormal expansion at the pluripotent stem cell level of a single clone of hemopoietic cells. Although it seems likely that this is related to the heightened proliferative activity characteristic of primitive CML progenitor cell types, the underlying mechanism is unknown. In this report we show that either normal or CML peripheral blood progenitors can be maintained on preestablished normal marrow adherent layers for periods of 1-2 months. Under these conditions numbers of both normal and neoplastic progenitors are usually higher in the adherent layer than in the nonadherent fraction. Moreover, the number of primitive progenitors of high proliferative potential present in the adherent layer is sufficient to allow their cycling status to be determined. Such measurements demonstrate that primitive normal progenitors of blood origin, when cultured in the presence of a preestablished adherent marrow feeder layer, go in and out of cycle after each medium change but in the absence of a feeder layer remain continuously in cycle. In contrast, primitive CML progenitors of either blood or marrow origin cycle continuously regardless of the presence or absence of an adherent feeder layer. We suggest that early expansion of the CML clone is related to an ability of the neoplastic stem cells to ignore or overcome a negative regulatory signal produced by nonneoplastic adherent marrow cells whose normal function is to maintain the stem cell reserve in a quiescent state.
Exchange of guanine nucleotide between GTP-binding proteins that regulate neuronal adenylate cyclase.
Authors: Hatta S , Marcus M M , Rasenick M M
Published: May 2006

GTP-binding proteins have been demonstrated to stimulate and inhibit rat brain adenylate cyclase without the prior addition of hormone. Exposure of rat cerebral cortex membranes to hydrolysis-resistant GTP analogs results in inhibition (or stimulation) of adenylate cyclase, which persists subsequent to buffer washing. The hydrolysis-resistant GTP photoaffinity probe P3-(4-azidoanilido)-P1-5' GTP (AAGTP) can promote a similar persistent inhibition of adenylate cyclase, and, after removal of unbound AAGTP and subsequent UV photolysis, AAGTP is covalently linked to the 40-kDa inhibitory GTP binding protein, GNi (inhibitory guanine nucleotide binding regulatory subunit of adenylate cyclase). Under conditions where the persistent inhibition of adenylate cyclase is overcome by subsequent incubation with 5'-guanylyl imidodiphosphate or NaF, AAGTP bound to the 40-kDa GNi protein is diminished while that bound to the 42-kDa stimulatory GTP-binding protein (GNs) is increased. Additionally, we have identified a 32-kDa protein that binds AAGTP with an affinity similar to that of GNs. This protein does not appear to be a byproduct of proteolysis as demonstrated by Staphylococcus aureus V8 protease digestion experiments, and it is not a substrate for ADP-ribosylation by bacterial toxins. The sum of the AAGTP bound by the GNi and GNs proteins is constant, and the transfer of nonphotoactivated AAGTP to GNs from GNi is stable to buffer washing. Furthermore, this alteration in the AAGTP-labeling pattern corresponds to the shift in adenylate cyclase from inhibition to stimulation. These data raise the possibility that hydrolysis-resistant GTP analogs might be exchanged directly between the GNi and GNs and that there exists some interaction between those proteins in the regulation of adenylate cyclase activity.
Differentiation-dependent sensitivity of human B-cell-derived lines to major histocompatibility complex-restricted T-cell cytotoxicity.
Authors: Torsteinsdottir S , Masucci M G , Ehlin-Henriksson B , Brautbar C , Ben Bassat H , Klein G , Klein E
Published: May 2006

Sets of Burkitt lymphoma lines and Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) derived from the same individuals were compared for sensitivity to cytotoxic T-lymphocyte (CTL) clones. Major histocompatibility complex class I antigen-restricted CTL clones were generated by stimulating the lymphocytes of an EBV-seropositive individual with the autologous LCL. One clone (BK-20) lysed the autologous and allogeneic HLA-A11-expressing LCLs but not mitogen-induced B lymphoblasts. Thus the clone was selectively cytotoxic for LCLs. Allospecific CTL clones directed against the HLA-A11 antigen were generated from an EBV-seronegative individual. One clone (WP-36) was selectively cytotoxic for the appropriate allospecific LCL, whereas another clone (WP-21) lysed also T and B lymphoblasts. None of the four Burkitt lymphoma lines established in parallel with the CTL-sensitive LCLs were lysed. Two of the Burkitt lymphoma lines were EBV-negative, and EBV-positive sublines were derived from these by in vitro infection. One but not the other of the two convertants became sensitive to all three types of CTL clones. The CTL-sensitive converted line had also acquired some LCL characteristics: increased cell size, aggregation, and a shift in several of the B-cell-specific surface markers. The CTL-resistant convertant expressed EBV antigens but showed no phenotypic change. These findings suggest that the cellular phenotype plays a decisive role in the sensitivity of B-cell-derived lines to the lytic effect of LCL-selective autologous and allogeneic CTLs.
A deletion in a rat major histocompatibility complex class I gene is linked to the absence of beta 2-microglobulin-containing serum molecules.
Authors: Björck L , Kryspin-Sörensen I , Dyrberg T , Lernmark A , Kastern W
Published: May 2006

Class I major histocompatibility antigens are composed of a heavy chain that is noncovalently associated with beta 2-microglobulin (beta 2m). Most class I molecules are membrane bound, but mouse and rat cDNA clones and genes without a functional code for the transmembrane amino acids have been identified. The membrane-associated class I molecules are important in the control of cell-mediated cytotoxicity, while the function of the soluble molecules remains unclear. Previous studies have shown that beta 2m circulates in rat serum in three different molecular weight classes. The first is free beta 2m (Mr, 12,000), the second is about Mr 70,000, and the third is roughly Mr 200,000. In an inbred subline of immunodeficient, diabetes-prone BioBreeding rats (BioBreeding/Hagedorn), previous work detected two restriction fragment polymorphisms in class I major histocompatibility complex genes, one of them a gene deletion on a 7-kilobase BamHI fragment and the other on a 2-kilobase BamHI fragment. In these rats we have found that the third serum beta 2m-binding size class is absent. Analysis of F1 and F2 individuals following cross-breeding between BioBreeding/Hagedorn rats and genetically related (nondiabetic) control BioBreeding w-subline rats demonstrated that the large-size serum peak of beta 2m was associated with the presence of the class I restriction fragments.
Identification of a partial cDNA clone for the C3d/Epstein-Barr virus receptor of human B lymphocytes: homology with the receptor for fragments C3b and C4b of the third and fourth components of complement.
Authors: Weis J J , Fearon D T , Klickstein L B , Wong W W , Richards S A , de Bruyn Kops A , Smith J A , Weis J H
Published: May 2006

Human complement receptor type 2 (CR2) is the B-lymphocyte receptor both for the C3d fragment of the third component of complement and for the Epstein-Barr virus. Amino acid sequence analysis of tryptic peptides of CR2 revealed a strong degree of homology with the human C3b/C4b receptor, CR1. This homology suggested that CR1 gene sequences could be used to detect the CR2 sequences at conditions of low-stringency hybridization. Upon screening a human tonsillar cDNA library with CR1 cDNA sequences, two clones were identified that hybridized at low, but not at high, stringency. Redundant oligonucleotides specific for CR2 sequences were synthesized and used to establish that the two cDNA clones weakly hybridizing with the CR1 cDNA contained CR2 sequences. One of these CR2 cDNA clones hybridized to oligonucleotides derived from two distinct CR2 tryptic peptides, whereas the other, smaller cDNA clone hybridized to oligonucleotides derived from only one of the CR2 peptides. Nucleotide sequence analysis of the CR2 cDNA confirmed that the site of oligonucleotide hybridization was identical to that predicted from the peptide sequence, including flanking sequences not included within the oligonucleotide probes. The CR2-specific cDNA sequences identified a poly(A)+ RNA species of 5 kilobases in RNA extracted from human B cells but did not hybridize to any RNA obtained from the CR2-negative T-cell line HSB-2, thus confirming the appropriate size and tissue-specific distribution for the CR2 mRNA. The striking peptide sequence homology between CR2 and CR1 and the cross-hybridization of the CR2 cDNA with the CR1-specific sequences allow the placement of CR2 in a recently defined gene family of C3- and C4-binding proteins consisting of CR1, C4-binding protein, factor H, and now, CR2.
From instability to irreversibility
Authors: Elskens Y. , Prigogine I.
Published: May 2006

A canonical procedure transforming the unitary evolution group

U
t

in a contracting semigroup

W
t

for phase-space ensembles has been developed for Kolmogorov dynamical systems in a series of recent papers. This paper investigates the physical meaning of this transformation. We stress that, for sufficiently unstable dynamical systems in which phase-space points are identified with an arbitrary but finite precision, one must take into account the undiscernibility of trajectories having the same asymptotic behavior in the future. The fundamental objects of our description are thus bundles of converging trajectories. We show that such an ensemble, corresponding to initial conditions whose support has finite measure, is then represented by a distribution function (called a Boltzmann ensemble) that evolves to equilibrium under the action of a markovian semigroup. The usual Gibbs-Koopman ensembles satisfying the Liouville equation are recovered as a singular limit. This work validates Boltzmann's intuition for a class of unstable dynamical systems and appears as a step toward the derivation of equations exhibiting irreversibility at a microscopic level.
Protein kinase C directly phosphorylates the insulin receptor in vitro and reduces its protein-tyrosine kinase activity.
Authors: Bollag G E , Roth R A , Beaudoin J , Mochly-Rosen D , Koshland D E
Published: May 2006

The beta subunit of purified insulin receptor is phosphorylated on a serine residue by purified preparations of protein kinase C (ATP: protein phosphotransferase, EC 2.7.1.37). This phosphorylation is inhibited by antibodies to protein kinase C and stimulated by phospholipids, diacylglycerol, and Ca2+. The phosphorylation of the receptor by protein kinase C does not affect its insulin-binding activity but does inhibit by 65% the receptor's intrinsic tyrosine-specific protein kinase activity (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112). These results indicate that activators of protein kinase C, such as phorbol esters, desensitize cells to insulin by direct protein kinase C action on the insulin receptor.
Activating mutations of the c-Ha-ras protooncogene in chemically induced hepatomas of the male B6C3 F1 mouse.
Authors: Wiseman R W , Stowers S J , Miller E C , Anderson M W , Miller J A
Published: May 2006

Activated c-Ha-ras protooncogenes have recently been identified in the DNA of some spontaneous hepatic tumors found in 2-year-old B6C3 F1 mice. Activation of c-Ha-ras has now been demonstrated in DNA from well-differentiated hepatomas initiated by a single dose of carcinogen given to male B6C3 F1 mice at 12 days of age. DNA from each of 25 hepatomas, induced by N-hydroxy-2-acetylaminofluorene, vinyl carbamate, or 1'-hydroxy-2',3'-dehydroestragole, containing transforming activity in the NIH 3T3 transfection assay. Southern analysis of NIH 3T3 cells transformed by DNA from 24 of these hepatomas revealed amplified and/or rear-ranged restriction fragments homologous to a Ha-ras probe. The other tumor contained an activated Ki-ras gene. Immunoprecipitation and NaDodSO4/PAGE analysis of p21 ras proteins in NIH 3T3 transformants derived from a majority of the hepatomas suggested that the activating mutations were localized in the 61st codon of the c-Ha-ras gene. Creation of a new Xba I restriction site by an AT----TA transversion at the second position of codon 61 was detected in DNA from primary tumors and NIH 3T3 cells transformed by DNA from 6 of 7 vinyl carbamate- and 5 of 10 1'-hydroxy-2',3'-dehydroestragole-induced hepatomas. Selective oligonucleotide hybridization demonstrated a CG----AT transversion at the first position of the 61st codon in NIH 3T3 transformants derived from 7 of 7 N-hydroxy-2-acetylaminofluorene-induced hepatomas. By the same criterion, an AT----GC transition at the second position of codon 61 was the activating mutation in 1 of 7 vinyl carbamate- and 5 of 10 1'-hydroxy-2',3'-dehydroestragole-induced tumors. Thus, c-Ha-ras activation is apparently an early event in B6C3 F1 mouse hepatocarcinogenesis that results directly from reaction of ultimate chemical carcinogens with this gene in vivo.
RNA splicing and intron turnover are greatly diminished by a mutant yeast branch point.
Authors: Jacquier A , Rosbash M
Published: May 2006

Two mutant genes, both of which contain an A----C transversion at the absolutely conserved branch point of the yeast "TACTAAC box" (TACTAAC----TACTACC), were constructed and introduced into yeast cells. Splicing and gene expression are almost completely eliminated by this mutation, but a low level (approximately equal to 0.1%) of proper splicing is detectable. Branch point mapping indicates that the mutant branch is formed at the normal location--i.e., to cytidine rather than adenosine. The mutant branch is also a very poor substrate for the HeLa cell debranching enzyme. Although splicing of the mutant transcripts is very poor, the cells contain a high level of mutant intron because these excised introns are remarkably stable. The results imply that the normal branch point is important not only for branch formation and splicing but also for intron turnover.
Treatment of human platelets with trypsin, thrombin, or collagen inhibits the pertussis toxin-induced ADP-ribosylation of a 41-kDa protein.
Authors: Lapetina E G , Reep B , Chang K J
Published: May 2006

Permeabilization of human platelets with saponin (15-25 micrograms/ml) allows the determination of the ADP-ribosylation of a 41-kDa protein by pertussis toxin. The ADP-ribosylated protein is present in the particulate fraction. ADP-ribosylation of the 41-kDa protein increases for 20 min; it is not affected by indomethacin, prostacyclin, and 1,2-diacylglycerols but is inhibited by 1 mM Ca2+ and phorbol esters. Treatment of platelets with trypsin, thrombin, or collagen before saponin addition precludes subsequent pertussis toxin-induced ADP-ribosylation of the 41-kDa protein. The effect of trypsin or thrombin is blocked by soybean trypsin inhibitor and leupeptin. Trypsin proteolytically cleaves the ADP-ribosylated 41-kDa protein to an ADP-ribosylated fragment slightly smaller than 20 kDa. The results suggest that a modification of a guanine nucleotide-binding regulatory protein is associated with the actions of trypsin, thrombin, and collagen on platelet activation.
Immunoglobulin heavy chain locus of the rat: striking homology to mouse antibody genes.
Authors: Brüggemann M , Free J , Diamond A , Howard J , Cobbold S , Waldmann H
Published: May 2006

DNA encoding the rat diversity segment (D), joining segment (JH), and constant (C) region mu, gamma 2a, gamma 1, gamma 2b, epsilon and alpha of the Ig heavy chain has been isolated from a cosmid library. Restriction mapping allowed us to identify two gene clusters: D-JH-C mu and C gamma 1-C gamma 2b-C epsilon-C alpha in addition to a single C gamma 2a gene. Analysis of genomic DNA by Southern blotting permitted identification of the C gamma 2c gene and led to the proposal of the following gene order for the rat Ig heavy chain locus: D-JH-C mu-C delta-(C gamma 2c, C gamma 2a)-C gamma 1-C gamma 2b-C epsilon-C alpha. There is striking homology between the rat and mouse Ig heavy chain loci as regards gene order and distance between CH genes. Partial DNA sequencing confirms this homology and shows that exon sequences are more conserved than are intron sequences. One of the most conserved intron regions between rat and mouse is that spanning the Ig heavy chain enhancer (91% homology). However, the relationship between the different C gamma subclasses in rat differs from that in mouse. Comparison of the C gamma CH3 domains shows that the rat C gamma 2b gene is most homologous to mouse C gamma 2a/b, whereas the rat C gamma 1 and C gamma 2a genes, both very similar to each other, are most homologous to the mouse C gamma 1 gene.
Expression of type C-related endogenous retroviral sequences in human colon tumors and colon cancer cell lines.
Authors: Gattoni-Celli S , Kirsch K , Kalled S , Isselbacher K J
Published: May 2006

Type C-related human endogenous retroviral sequences have been previously discovered and characterized [Martin, M. A., Bryan, T., Rasheed, S. & Khan, A.S. (1981) Proc. Natl. Acad. Sci. USA 78, 4892-4896]. We investigated the transcriptional pattern of these sequences to determine whether and to what extent their expression is altered in colon tumors and colon cancer cell lines as compared to normal colon mucosa (NCM). Of two long terminal repeat (LTR)-specific transcripts [3.6 and 2.1 kilobases (kb)], the 3.6-kb RNA was particularly abundant in NCM but strikingly decreased in most primary colon cancers tested. In NCM we identified three envelope gene (env)-related transcripts--namely, 3.0, 1.7, and 0.6 kb. Colon tumors appeared to express higher levels of these transcripts, especially the 1.7-kb species. In one case of dysplasia and in one benign tumor, this 1.7-kb transcript was clearly increased. We also examined the pattern of transcription in colon cancer cell lines HCT and Caco2. The LTR-homologous 3.6-kb transcript, very abundant in NCM, was decreased in primary tumors and in HCT cells and virtually absent in Caco2 cells. The latter, however, appeared to produce the transcript when growing exponentially, indicating that Caco2 cultures provide an inducible system susceptible to in vitro manipulation. Both cell lines also contained higher amounts of the 1.7-kb env-related transcript. The decrease of the 3.6-kb RNA in colon tumors versus NCM may be the result of an altered pattern of differentiation, whereas the increase of the 1.7-kb RNA in tumors may represent an early marker of colon neoplasia.
Calcium and calmodulin-enhanced in vitro phosphorylation of hen brain cold-stable microtubules and spinal cord neurofilament triplet proteins after a single oral dose of tri-o-cresyl phosphate.
Authors: Suwita E , Lapadula D M , Abou-Donia M B
Published: May 2006

The effect of a single 750-mg/kg oral dose of tri-o-cresyl phosphate (TOCP) on the endogenous phosphorylation of brain microtubule preparations and spinal cord neurofilaments was assessed in hens after the development of delayed neurotoxicity. Protein phosphorylation with [gamma-32P]ATP was analyzed by one-dimensional and two-dimensional gel electrophoresis, autoradiography, and microdensitometry. TOCP treatment enhanced the Ca2+- and calmodulin-dependent phosphorylation of tubulin in crude chicken brain cytosol (160% for alpha-tubulin and 140% for beta-tubulin) and cold-stable microtubules (165% and 155% for alpha- and beta-tubulin, respectively). Microtubule-associated protein 2 (MAP-2) phosphorylation was also increased in brain fractions studied--i.e., brain cytosol (145%), cold-stable microtubules (133%), and cold-labile microtubules (328%). There was significant increase in phosphorylation of a 70-kDa protein in the brain cytosol and in the cold-stable microtubule fractions. TOCP also stimulated the phosphorylation of spinal cord proteins of 70 kDa (119%) and 160 kDa (129%) in a Mg2+-dependent manner. Addition of Ca2+ and calmodulin further enhanced the phosphorylation of these 70-kDa (563%) and 160-kDa (221%) proteins as well as of 52-, 59-, and 210-kDa proteins by as much as 126%, 160%, and 196%, respectively. Two-dimensional electrophoresis was carried out to identify these proteins. They were confirmed as alpha- and beta-tubulin (52 and 59 kDa) in brain and spinal cord preparations and the neurofilament triplet proteins (70, 160, and 210 kDa) in the spinal cord preparation. The 70-kDa protein in brain was not neurofilament in origin. Peptide mapping using Staphylococcus aureus V8 protease showed the brain and spinal cord cytoskeletal proteins have identical phosphopeptide patterns in control and TOCP-treated hens, indicating that it was unlikely that the phosphorylation sites were altered by TOCP treatment.
Ethanol regulates calcium channels in clonal neural cells.
Authors: Messing R O , Carpenter C L , Diamond I , Greenberg D A
Published: May 2006

The acute and long-term effects of ethanol on voltage-dependent Ca channel function were studied in PC12, a clonal cell line of neural crest origin. Acute exposure to ethanol produced a concentration-dependent decrease in depolarization-evoked 45Ca2+ uptake, while prolonged (2-10 days) exposure led to a reciprocal increase in 45Ca2+ uptake and in the number of Ca-channel binding sites labeled by the dihydropyridine Ca-channel antagonist [3H]nitrendipine. Uptake was restored to control levels following withdrawal of ethanol from cultures. These findings indicate that cellular adaptation to ethanol may involve enhanced expression of dihydropyridine-sensitive, voltage-dependent Ca channels.
Fluorescent actin filaments move on myosin fixed to a glass surface.
Authors: Kron S J , Spudich J A
Published: May 2006

Single actin filaments stabilized with fluorescent phalloidin exhibit ATP-dependent movement on myosin filaments fixed to a surface. At pH 7.4 and 24 degrees C, the rates of movement average 3-4 micron/s with skeletal muscle myosin and 1-2 micron/s with Dictyostelium myosin. These rates are very similar to those measured in our previous myosin movement assays. The rates of movement are relatively independent of the type of actin used. The filament velocity shows a broad pH optimum between 7.0 and 9.0, and the concentration of ATP required for half-maximal velocity is 50 microM. Evidence was obtained to suggest that movement of actin over myosin requires at most the number of heads in a single thick filament. This system provides a practical, quantitative myosin-movement assay with purified proteins.
Functional expression of rat cytochrome c in Saccharomyces cerevisiae.
Authors: Scarpulla R C , Nye S H
Published: May 2006

To determine whether a mammalian cytochrome c could efficiently replace iso-1-cytochrome c, which is encoded by the yeast CYC1 gene, the coding sequence of RC9 (a nondefective processed gene from rat) was cloned in both single- and multiple-copy expression vectors under the direction of the yeast alcohol dehydrogenase 1 (ADC1) promoter. Upon transformation of a CYC1 deletion strain, the multiple-copy construct restored a wild-type growth rate on lactate medium; such growth normally requires derepressed amounts of iso-1-cytochrome c. These transformants expressed a level of hybrid ADC1/RC9 mRNA approximately 5- to 10-fold greater than the amount of message from the endogenous ADC1 gene and produced a steady-state level of rat cytochrome c equivalent to that of the wild-type yeast protein. A requirement for the vector was evidenced by its absence in all transformants that lost the lactate growth phenotype after propagation in nonselective medium. In contrast, the level of vector-specific message in single copy was equivalent to that of the endogenous ADC1 mRNA, but transformants exhibited no significant growth on lactate. Constructions having a small deletion or a mammalian intron within the rat cytochrome c coding region failed to support lactate-dependent growth, indicating that complementation depends upon proper translation of the correct rat coding sequence. Therefore, the rat polypeptide, when expressed at normal physiological levels, is recognized by the yeast machinery involved in the multiple steps required for the processing and transport of an active cytochrome c as well as its functional interaction with the respiratory apparatus.
Phosphorylation of the fibronectin receptor complex in cells transformed by oncogenes that encode tyrosine kinases.
Authors: Hirst R , Horwitz A , Buck C , Rohrschneider L
Published: May 2006

The fibronectin (FN) receptor in avian cells has been characterized previously as a complex of three membrane glycoproteins of about Mr 160,000, Mr 140,000, and Mr 120,000 (simply termed protein band 1, band 2, and band 3, respectively). Monoclonal antibodies to the band 3 protein of the complex prevent FN and laminin binding both in vivo and in vitro and enable the detection of the receptor proteins in the plasma membrane and in adhesion plaques. Association of the FN receptor proteins with the adhesion-plaque protein talin also has been reported. We now find that the band 2 and band 3 proteins in the complex are phosphorylated in Rous sarcoma virus-transformed chicken cells but not in normal chicken cells. Phosphorylation occurs predominantly on tyrosine and is accompanied by a reorganization of the receptor complex in the membrane of the transformed cells. Whereas normal cells contain the FN receptor in focal contacts and cellular processes between cells, v-src-transformed cells exhibit a more diffuse distribution of this receptor. In addition to the viral v-src oncogene, cells transformed by other avian oncogenes that also encode tyrosine kinases (v-fps, v-erbB, and v-yes) also express the receptor complex proteins in the phosphorylated state regardless of whether the transforming protein is detectable in adhesion plaques. These results suggest that the altered FN and laminin receptor proteins may contribute to the transformed phenotype, but their significance and role in the transformed state remain to be established.
Cloning of cDNA and amino acid sequence of a cytokeratin expressed in oocytes of Xenopus laevis.
Authors: Franz J K , Franke W W
Published: May 2006

Using a cDNA clone from the ovary of the frog, Xenopus laevis, we have identified the mRNA and determined the complete amino acid sequence of a major cytoskeletal protein expressed in the oocyte. A comparison with other cytoskeletal proteins of Xenopus and mammals identifies this polypeptide Mr 55,700 as a nonepidermal kind of cytokeratin of the basic (type II) subfamily, which represents the amphibian equivalent to cytokeratin no. 8 of simple epithelia of higher mammals. The sequence data demonstrate the high evolutionary stability of this protein. This cytokeratin and its mRNA are present in oocytes, eggs, embryos, liver, and intestinal mucosa of adult frogs, as well as cultured kidney epithelial cells. We suggest that epithelial cell differentiation in early stages of Xenopus embryogenesis differs from other known pathways of cell differentiation in that major cell-type-specific proteins--i.e., cytokeratins of the simple epithelial type--and their mRNAs are maternally provided and distributed to early epithelial cells by special sorting mechanisms.
Carrier-directed targeting of liposomes and erythrocytes to denuded areas of vessel wall.
Authors: Smirnov V N , Domogatsky S P , Dolgov V V , Hvatov V B , Klibanov A L , Koteliansky V E , Muzykantov V R , Repin V S , Samokhin G P , Shekhonin B V
Published: May 2006

Immunomorphological staining of specimens prepared from human carotid arteries with anti-collagen type I antibodies reveals large amounts of type I collagen in the subendothelium of lipid fibrous plaques. Collagen type I-containing structures, once in direct contact with blood after plaque rupture, can serve as potential targets for selective delivery of liposomes and erythrocytes to these areas. To verify this rationale, [14C]cholesterol oleate-containing liposomes were conjugated with bovine or human anti-collagen type I antibodies or human plasma fibronectin. Biotin derivatives of human anti-collagen type I antibody were coupled to human erythrocytes. Modified liposomes and erythrocytes were perfused in situ through segments of bovine, rabbit, or human arteries partially denuded with a balloon catheter prior to perfusion. After perfusion, the control and denuded areas were excised and subjected to scanning electron microscopic analysis and measurements of associated radioactivity. It was found that conjugates of liposomes or erythrocytes with anti-collagen type I antibodies or fibronectin are selectively bound by endothelium-free zones of arterial segments. Carrier-directed targeting of drug-laden liposomes and erythrocytes to thrombosis-prone areas of arterial lumen is discussed.
Antitumor effects of an immunotoxin made with Pseudomonas exotoxin in a nude mouse model of human ovarian cancer.
Authors: FitzGerald D J , Willingham M C , Pastan I
Published: May 2006

An immunotoxin composed of Pseudomonas toxin coupled to an antibody to the human transferrin receptor was evaluated for its effect on ovarian cancer. In the tumor model employed, 60 million human ovarian cancer cells were injected into the peritoneal cavity of an immunodeficient nude mouse. By day 5, cancer cells were implanted and growing in small clusters throughout the peritoneal cavity. On days 5-8, 0.3-2 micrograms of immunotoxin was injected into the peritoneal cavity. Control mice died with malignant ascites at 34-58 days after the implantation of tumor cells, whereas immunotoxin-treated mice lived to 100 days or longer. Irrelevant immunotoxins or antibody alone had no antitumor activity. These findings suggest that intraperitoneal injection of immunotoxins may have a role in the treatment of ovarian cancer.
Ca2+- and K+-dependent communication between central nervous system myelinated axons and oligodendrocytes revealed by voltage-sensitive dyes.
Authors: Lev-Ram V , Grinvald A
Published: May 2006

The interactions between myelinated axons and surrounding glia cells, in rat optic nerve, were investigated by optical recording with voltage-sensitive dyes. Electrical stimulation of the nerve evoked an optical signal revealing two clearly distinct components: a fast propagating component, corresponding to the compound action potential, and a prominent slow component. Several lines of evidence suggest that part of the slow component originated from depolarization of the oligodendrocytes by potassium accumulation in the paranodal or internodal region. In addition, the experiments suggest that in this preparation axons also have voltage-dependent Ca2+ channels, and a Ca2+-dependent K+ conductance involved in the depolarization of oligodendrocytes. Thus, axons and oligodendrocytes communicate in an intimate, ionically-mediated fashion, and oligodendrocytes may play an important functional role beyond that of providing the myelin sheath.
Existence of simply connected algebraic surfaces of general type with positive and zero indices
Authors: Moishezon Boris , Teicher Mina
Published: May 2006

In the classification problem of algebraic surfaces of general type, an important conjecture states that for simply connected such surfaces Chern numbers satisfy the inequality
c
1
2
≤ 2
c
2
(or equivalently, the index τ ≤ 0). We disprove this conjecture by computing fundamental groups of Galois coverings corresponding to generic C
P
2
projections of projective embeddings of C
P
1
× C
P
1
related to linear systems [unk]
al
1
+
bl
2
[unk],
a
≥ 3,
b
≥ 2. Also, we proved the existence of simply connected minimal surfaces of general type with zero index (e.g.,
c
1
2
= 2
c
2
). Previously, it was conjectured that these are exactly the surfaces uniformizable in the polydisk. So this conjecture is also disproved.
Human cytochrome P-450 4 mRNA and gene: part of a multigene family that contains Alu sequences in its mRNA.
Authors: Quattrochi L C , Pendurthi U R , Okino S T , Potenza C , Tukey R H
Published: May 2006

Several overlapping lambda gt11 cDNA clones have been sequenced and shown to encode for the full-length human cytochrome P-450 4. The structure and location of the exons and flanking intron regions were also identified from a lambda EMBL-3 human genomic clone that encodes the full-length human P-450 4 gene. The human P-450 4 mRNA is flanked by 62 base pairs of 5'- and 1508 base pairs of 3'-noncoding sequence, with 1548 bases that encode a protein of 516 amino acids (Mr, 58,376). The predicted amino acid sequence of human P-450 4 is 69% and 70% homologous to its equivalent in mouse and rat, respectively, 75% homologous to rabbit P-450 4, and 68% homologous to human P1-450. The 7.6-kilobase gene encodes 3118 nucleotides of exon sequence that is separated by six introns into seven exons. Exon 7, which is 1802 nucleotides, contains three inverse/complement Alu sequences that are organized in tandem. Comparison of the genomic DNA sequence of the human P-450 4 gene with the human P1-450 and related genes in rat and mouse and the identification of the amino acid residues and triplet codon at each exon-intron junction show that the location of each intron in the human P-450 4 gene is conserved within this gene family. Although the length and homology of the introns within a related gene family may not be conserved, the location of intronic sequences may be an important determinant in the identification of related P-450 genes.
Molecular cloning and nucleotide sequence of a full-length cDNA for human alpha enolase.
Authors: Giallongo A , Feo S , Moore R , Croce C M , Showe L C
Published: May 2006

We previously purified a 48-kDa protein (p48) that specifically reacts with an antiserum directed against the 12 carboxyl-terminal amino acids of the c-myc gene product. Using an antiserum directed against the purified p48, we have cloned a cDNA from a human expression library. This cDNA hybrid-selects an mRNA that translates to a 48-kDa protein that specifically reacts with anti-p48 serum. We have isolated a full-length cDNA that encodes p48 and spans 1755 bases. The coding region is 1299 bases long; 94 bases are 5' noncoding and 359 bases are 3' noncoding. The cDNA encodes a 433 amino acid protein that is 67% homologous to yeast enolase and 94% homologous to the rat non-neuronal enolase. The purified protein has been shown to have enolase activity and has been identified to be of the alpha type by isoenzyme analysis. The transcriptional regulation of enolase expression in response to mitogenic stimulation of peripheral blood lymphocytes and in response to heat shock is also discussed.
Rat skeletal myoblasts and arterial smooth muscle cells express the gene for the A chain but not the gene for the B chain (c-sis) of platelet-derived growth factor (PDGF) and produce a PDGF-like protein.
Authors: Sejersen T , Betsholtz C , Sjölund M , Heldin C H , Westermark B , Thyberg J
Published: May 2006

It is shown here that the myogenic cell line L6J1, primary skeletal myoblasts, and primary adult arterial smooth muscle cells express the gene for the A chain but not the gene for the B chain (c-sis) of platelet-derived growth factor (PDGF). It is further demonstrated that conditioned media from L6J1 cultures contain material that (i) competes with 125I-labeled PDGF for binding to human fibroblasts, (ii) is specifically precipitated by antibodies against PDGF, and (iii) has a relative molecular mass comparable to that of PDGF and, after reduction, its constituent subunit chains. The secretion of PDGF-receptor-competing activity was at a maximum in exponentially growing cultures but remained at a high level also after the cells had become confluent, stopped dividing, and fused to form multinucleate myotubes. Similarly, it was previously demonstrated that adult rat arterial smooth muscle cells in primary culture produce a mitogenic protein with immunological and structural properties similar to PDGF. In accordance with these findings, it was recently shown that secretion of PDGF-like mitogens by a number of human tumor cell lines correlates with expression of the gene for the A chain rather than the B chain of PDGF. The results suggest that production of homodimers of PDGF A chains may stimulate proliferation of skeletal myoblasts and arterial smooth muscle cells in an autocrine or paracrine manner. This could fulfill important functions during myogenesis in the embryo as well as in tissue repair and atherogenesis in the adult.
Human fibroblasts accelerate the inhibition of thrombin by protease nexin.
Authors: Farrell D H , Cunningham D D
Published: May 2006

Protease nexin (PN) is a protein protease inhibitor secreted by human fibroblasts in culture that complexes and inhibits certain regulatory serine proteases. The PN-protease complexes then bind to these cells and are rapidly internalized and degraded. This report shows that the fibroblast surface accelerates the formation of PN-thrombin complexes. In contrast, it did not accelerate the formation of complexes between thrombin and antithrombin III, a closely related protease inhibitor found in plasma. These results support a role for PN in the regulation of certain proteases in the extravascular compartment at and near the surface of tissue cells. The activity that accelerated PN-thrombin complex formation was membrane-associated, since fixed cells, purified membranes, and extracellular matrix preparations all contained this activity. The ability of cells to accelerate the reaction between PN and thrombin was inhibited by protamine, suggesting that the activity was similar to that of heparin. Heparitinase digestion of plasma membranes prior to assay reduced the activity by about 80%, suggesting that heparan sulfate may account for most of the accelerative activity.
Role of calcium in regulating the cyclic GMP cascade of phototransduction in retinal rods.
Authors: Torre V , Matthews H R , Lamb T D
Published: May 2006

Both cGMP and Ca2+ appear to be involved in the process of phototransduction in vertebrate rods, but their precise roles have been the subject of debate. To investigate the role of Ca2+ we have artificially increased the calcium buffering capacity of the rod by using a patch pipet to incorporate the calcium buffer 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) into the rod cytoplasm. In the presence of buffer the Na+-Ca2+ exchange current became greatly slowed, suggesting that the cytoplasmic calcium concentration (Cai) had indeed been buffered substantially. Although the presence of buffer had negligible effect on the rising phase of the light response, it profoundly altered the later behavior. Responses to brief flashes became prolonged and exhibited an overshoot, apparently because the shut-off process was modified. The normal acceleration of time-to-peak with brighter flashes (an early sign of light adaptation) disappeared. Responses to steady adapting illumination took much longer than normal to settle to a steady level, although the final level represented a similar fractional suppression of current. With superimposed test flashes the presence of such adapting illumination caused a more rapid recovery, whereas the presence of calcium buffer slowed the recovery. The results are consistent with the idea that the rapid drop in Cai, which has recently been shown to accompany the light response, is involved in terminating the light response, and that Cai is thereby involved in setting the operating point and sensitivity of phototransduction. From comparison with other work we infer that Cai appears to act, at least in part, by means of control of cGMP phosphodiesterase activity.
Structure of evolving populations of Saccharomyces cerevisiae: adaptive changes are frequently associated with sequence alterations involving mobile elements belonging to the Ty family.
Authors: Adams J , Oeller P W
Published: May 2006

Haploid a and diploid a/alpha and a/a populations of Saccharomyces cerevisiae evolving in laboratory environments for up to 300 generations were analyzed for sequence rearrangements associated with the Ty family of transposable elements. In contrast to results with Escherichia coli, evolving populations of yeast exhibit a high frequency of sequence rearrangements associated with mobile genetic elements. In particular, adaptive shifts in these populations are often associated with such sequence rearrangements. The results are most compatible with the explanation that there is direct selection for some of the sequence rearrangements. In addition, the pattern of changes suggests that the structure of evolving microorganism populations may be more complex than expected.
Mitochondrial DNA changes in abnormal growth (nonchromosomal stripe) mutants of maize
Authors: Newton Kathleen J. , Coe E. H.
Published: May 2006

The genetic analysis of higher plant mitochondria has been limited by a scarcity of identified mutations with known progenitors. Correspondingly, few molecular studies have been directed at types of plant mitochondrial variation other than cytoplasmic male sterility. The maternally inherited nonchromosomal stripe (NCS) mutants of maize have profound deleterious effects on plant growth and yield. We report specific alterations in mitochondrial DNA (mtDNA) for two independent, phenotypically distinct NCS mutants. NCS2 plants have a distinctive 21-kilobase
Xho
I mtDNA band and very reduced amounts of DNA in an 8-kilobase band that is present in the progenitor. NCS3 plants have a distinctive 20-kilobase
Xho
I band and a reduction in a 16-kilobase band. Our studies confirm that the affected organelle in NCS plants is the mitochondrion. Because NCS-type plants appear with a certain frequency in a particular line (WF9), this line is a potential source of additional mutations for functional and molecular analyses of maize mitochondrial genes.