bluuebunny/crossref_metadata_2025 huggingface dataset:
Construction of the physical map for three loci in chromosome band 13q14: comparison to the genetic map.
Authors: Higgins M J , Turmel C , Noolandi J , Neumann P E , Lalande M
Published: May 2006

Pulsed-field gel electrophoresis (PFGE) and deletion mapping are being used to construct a physical map of the long arm of human chromosome 13. The present study reports a 2700-kilobase (kb) Not I long-range restriction map encompassing the 13q14-specific loci D13S10, D13S21, and D13S22, which are detected by the cloned DNA markers p7D2, pG24E2.4, and pG14E1.9, respectively. Analysis of a panel of seven cell lines that showed differential methylation at a Not I site between D13S10 and D13S21 proved physical linkage of the two loci to the same 875-kb Not I fragment. D13S22 mapped to a different Not I fragment, precluding the possibility that D13S22 is located between D13S10 and D13S21. PFGE analysis of Not I partial digests placed the 1850-kb Not I fragment containing D13S22 immediately adjacent to the 875-kb fragment containing the other two loci. The proximal rearrangement breakpoint in a cell line carrying a del13(q14.1q21.2) was detected by D13S21 but not by D13S10, demonstrating that D13S21 lies proximal to D13S10. Quantitative analysis of hybridization signals of the three DNA probes to DNA from the same cell line indicated that only D13S10 was deleted, establishing the order of these loci to be cen-D13S22-D13S21-D13S10-tel. Surprisingly, this order was estimated to be 35,000 times less likely than that favored by genetic linkage analysis.
Synergistic transcriptional enhancement does not depend on the number of acidic activation domains bound to the promoter.
Authors: Oliviero S , Struhl K
Published: May 2006

Many eukaryotic transcriptional activator proteins contain a DNA-binding domain that interacts with specific promoter sequences and an acidic activation region that is required to stimulate transcription. Transcriptional enhancement by such activator proteins is often synergistic and promiscuous; promoters containing multiple binding sites for an individual protein or even for unrelated proteins can be 10-100 times more active than promoters with single sites. It has been suggested that such synergy reflects a nonlinear response of the basic transcription machinery to the number and/or quality of acidic activation regions. Here, we determine the transcriptional activity of Jun-Fos heterodimers containing one or two GCN4 acidic activation regions on promoters containing one or two Ap-1 target sites. Surprisingly, heterodimers with one or two acidic regions activate transcription with similar efficiency and are equally synergistic (10- to 15-fold) on promoters containing two target sites. Thus, transcriptional synergy does not depend on the number of acidic activation regions but rather on the number of proteins bound to the promoter. This suggests that synergy is mediated either by cooperative DNA binding or by alternative mechanisms in which the DNA-binding domain plays a more direct role in transcription (e.g., changes in DNA structure, nucleosome displacement, or direct interactions with the transcriptional machinery).
A potential Z-DNA-forming sequence is located between two transcription units alternatively expressed during development of Drosophila hydei.
Authors: Jimenez-Ruiz A , Requena J M , Lopez M C , Alonso C
Published: May 2006

Recent studies have demonstrated that Z-DNA exists in vivo in Escherichia coli as well as in Drosophila and mammalian cells. In the present paper, we show the existence in vivo of Z-DNA epitopes in the developmentally regulated subregion 4-75C of polytene chromosomes in Drosophila hydei. The Z-DNA epitopes were detected in subdivision C2 only during late third instar when the transcriptional activity of the locus was high. Accumulation of nonhistone chromosomal proteins in that locus was also detected during late third instar only at the time of the Z-DNA formation. Northern blot data and nucleotide sequence analysis indicated that the Z-DNA-forming sequence is located between two transcription units whose expression is regulated during the third instar. Our results suggest that in subdivision 4-75C2 a B- to Z-DNA flux occurs at a specific time during late third instar and that this flux may play a negative as well as a positive role in gene expression.
Additional fossil evidence on the differentiation of the earliest euprimates.
Authors: Rose K D , Bown T M
Published: May 2006

Several well-preserved jaws of the rare North American omomyid primate Steinius vespertinus, including the first known antemolar dentitions, have been discovered in 1989 and 1990 in the early Eocene Willwood Formation of the Bighorn Basin, Wyoming. They indicate that its dental formula is as primitive as those in early Eocene Donrussellia (Adapidae) and Teilhardina (Omomyidae)--widely considered to be the most primitive known euprimates--and that in various dental characters Steinius is as primitive or more so than Teilhardina. Therefore, despite its occurrence at least 2 million years later than Teilhardina, S. vespertinus is the most primitive known omomyid and one of the most primitive known euprimates. Its primitive morphology further diminishes the dental distinctions between Omomyidae and Adapidae at the beginning of the euprimate radiation
Effects of metal ions, including Mg2+ and lanthanides, on the cleavage of ribonucleotides and RNA model compounds.
Authors: Breslow R , Huang D L
Published: May 2006

The cyclization/cleavage of 3',5'-uridyluridine to form 2',3'-cyclic uridylic acid is very effectively catalyzed by Eu3+, and the cyclization/cleavage of the 1-p-nitrophenyl phosphate ester of propane-1,2-diol also shows strong metal ion catalysis by Eu3+, Tb3+, and Yb3+. It also shows moderate catalysis by Mg2+, but not by Ca2+; Zn2+ and Pb2+ are also good catalysts. Various ligands activate these reactions further, and imidazole apparently acts as an additional base catalyst. Some cyclodextrin derivatives act to bind both the substrate and the metal ion but, contrary to what is reported elsewhere, there is no strong selectivity among nucleotides that can be ascribed to cyclodextrin binding.
Functional domains of the poliovirus receptor.
Authors: Koike S , Ise I , Nomoto A
Published: May 2006

A number of mutant cDNAs of the human poliovirus receptor were constructed to identify essential regions of the molecule as the receptor. All mutant cDNAs carrying the sequence coding for the entire N-terminal immunoglobulin-like domain (domain I) confer permissiveness for poliovirus to mouse L cells, but a mutant cDNA lacking the sequence for domain I does not. The transformants permissive for poliovirus were able to bind the virus and were also recognized by monoclonal antibody D171, which competes with poliovirus for the cellular receptor. These results strongly suggest that the poliovirus binding site resides in domain I of the receptor. Mutant cDNAs for the sequence encoding the intracellular peptide were also constructed and expressed in mouse L cells. Susceptibility of these cells to poliovirus revealed that the entire putative cytoplasmic domain is not essential for virus infection. Thus, the cytoplasmic domain of the molecule appears not to play a role in the penetration of poliovirus.
Gap junction protein homologue from Arabidopsis thaliana: evidence for connexins in plants.
Authors: Meiners S , Xu A , Schindler M
Published: May 2006

An Arabidopsis thaliana library constructed in the pBluescript expression vector lambda ZAP II (Stratagene) was screened with three affinity-purified antibodies raised against (i) rat liver connexin 32, (ii) a polypeptide from soybean root cells that migrates with a molecular mass of 29 kDa in SDS/polyacrylamide gels and is immunologically related to rat liver connexin, and (iii) a synthetic peptide corresponding to a sequence in rat liver connexin 32. A single clone was obtained whose gene product demonstrated immunological crossreactivity with all three reagents. The cDNA from this clone contained 1171 base pairs and coded for a protein of 280 amino acids with a calculated molecular mass of 32,339 Da (migrates as a 29-kDa polypeptide in SDS/polyacrylamide gels). The sequence homology observed between the 32-kDa polypeptide of Arabidopsis and connexin 32 from rat liver, in conjunction with observed similarities in predicted number and distribution of hydrophobic domains, sites for posttranslational modification, and basic pI, provides strong evidence that the biological range for connexin-type proteins may now be considered to include the plant kingdom.
Localization of receptors for luteinizing hormone-releasing hormone in pancreatic and mammary cancer cells.
Authors: Szende B , Srkalovic G , Timar J , Mulchahey J J , Neill J D , Lapis K , Csikos A , Szepeshazi K , Schally A V
Published: May 2006

Previous work showed that hamster and human pancreatic tumors but not normal pancreata exhibit low-affinity cell-membrane receptors for luteinizing hormone-releasing hormone (LHRH). Although the regression of experimental pancreatic cancers induced by treatment with LHRH agonists or antagonists could be explained in part by the creation of sex-steroid deficiency, direct effects mediated by LHRH receptors might also play a role. Here, we demonstrate that pancreatic tumor cells do exhibit high-affinity binding sites for LHRH, but only in their nuclei; low-affinity sites are associated with the cell membranes. These binding sites appear to be LHRH receptors since electron microscopic immunohistochemical studies show that an antibody to the LHRH receptor reacted with sites in the nucleus of pancreatic tumor cells. Immunoreactive sites in the nucleus also were found in a restricted set of normal hamster pituitary cells thought to be luteinizing hormone-secreting cells and in MXT mouse mammary tumor cells. Such nuclear receptors may be involved in the transmission of the direct action of LHRH analogues on the tumor cells, resulting in the enhancement of programmed cell death.
Protein geranylgeranyltransferase of Saccharomyces cerevisiae is specific for Cys-Xaa-Xaa-Leu motif proteins and requires the CDC43 gene product but not the DPR1 gene product.
Authors: Finegold A A , Johnson D I , Farnsworth C C , Gelb M H , Judd S R , Glomset J A , Tamanoi F
Published: May 2006

Protein prenylation occurs by modification of proteins with one of at least two isoprenoids, the farnesyl group and the geranylgeranyl group. Protein farnesyltransferases have been identified, but no such enzyme has been identified for geranylgeranylation. We report the identification of an activity in crude soluble yeast extracts that catalyzes the transfer of a geranylgeranyl moiety from geranylgeranyl pyrophosphate to proteins having the C-terminal sequence Cys-Ile-Ile-Leu or Cys-Val-Leu-Leu but not to a similar protein ending with Cys-Ile-Ile-Ser. This activity is dependent upon the CDC43/CAL1 gene, which is involved in budding and the control of cell polarity, but does not require the DPR1/RAM1 gene, which is known to be required for the farnesylation of Ras proteins. These results indicate that the protein geranylgeranyltransferase activity is distinct from the protein farnesyltransferase activity and that its specificity depends in part on the extreme C-terminal leucine in the protein to be prenylated.
Glycosylation site binding protein and protein disulfide isomerase are identical and essential for cell viability in yeast.
Authors: LaMantia M , Miura T , Tachikawa H , Kaplan H A , Lennarz W J , Mizunaga T
Published: May 2006

Glycosylation site binding protein (GSBP) has been shown to be identical to protein disulfide isomerase (PDI; EC 5.3.4.1) in a variety of multicellular organisms. We have utilized immunological and biochemical techniques to determine if GSBP and PDI are identical in yeast. Antiserum prepared against yeast GSBP identified in microsomes by its ability to be labeled with a peptide photoaffinity probe was found to recognize PDI purified from yeast. Moreover, this purified yeast PDI was found to be specifically labeled by the photoaffinity probe originally used to identify GSBP in a variety of eukaryotes. On the basis of these observations, we conclude that yeast GSBP and PDI are the same protein. The structure of the yeast PDI gene revealed a product with sequence similarity to higher eukaryotic PDI/GSBP. Disruption of this gene in yeast resulted in a recessive lethal mutation, indicating that PDI/GSBP is required for cell viability.
Human immunodeficiency virus infection of human lymph nodes in the SCID-hu mouse.
Authors: Kaneshima H , Shih C C , Namikawa R , Rabin L , Outzen H , Machado S G , McCune J M
Published: May 2006

The SCID-hu mouse is a small animal in which human hematolymphoid organs can be engrafted and maintained in vivo. In this study, parameters are described for reproducible infection of SCID-hu mice after i.v. inoculation. Infection was found to be dependent upon the time after inoculation, the virus isolate, the titer of virus, and the human target organ implanted into the mouse. Ten to 14 days after the i.v. administration of HIV isolates derived freshly from patients (e.g., JR-CSF, JR-FL, SM), 100% of engrafted human lymph nodes in SCID-hu mice were infected; greater than 95% of these animals were also viremic. Implants of human thymus or connective tissue, as well as the endogenous murine hematolymphoid organs, were not infected. As demonstrated by a combination of in situ hybridization and immunohistochemistry, both T-lymphoid and myelomonocytic lineage cells were infected in this system. HIV isolates that have been adapted to growth in vitro (e.g., HTLV-IIIb) were not infectious. When either 3'-azido-3'-deoxythymidine (AZT) or 2',3'-dideoxyinosine (ddIno) was administered to SCID-hu mice before HIV infection, the animals were protected in dose ranges similar to those used in man. This animal model may now be used as an efficient intermediate step between the lab and the clinic to study the infectious process in vivo and to best select efficacious antiviral compounds against HIV.
Cytolytic and ion channel-forming properties of the N terminus of lymphocyte perforin.
Authors: Ojcius D M , Persechini P M , Zheng L M , Notaroberto P C , Adeodato S C , Young J D
Published: May 2006

Perforin lyses cells by binding to the target cell membrane, where it polymerizes into large nonspecific pores. It is shown here that the first 34 amino acids of the N-terminal region of either human or murine perforin are soluble in aqueous medium and spontaneously insert into membranes. The N-terminal peptides lyse liposomes and nucleated cells, and they form ion channels in planar bilayers, some of which are comparable to those previously described for perforin. The lytic activity of the N-terminal domains does not require calcium, is independent of the lipid headgroup composition, and can be inhibited by heparin. Tumor cells incubated with the N-terminal peptides undergo the same morphological changes as those induced by native perforin. None of the peptides corresponding to the putative membrane-spanning domains from the central region of perforin is cytolytic. Taken together, these results suggest that the N-terminal region is an important part of the pore-forming domain of perforin.
A covalent complex between retroviral integrase and nicked substrate DNA.
Authors: Katzman M , Mack J P , Skalka A M , Leis J
Published: May 2006

Purified retroviral integrase (IN) from avian sarcoma-leukosis viruses can appropriately process the termini of linear viral DNA, cleave host DNA in a sequence-independent manner, and catalyze integrative recombination; an exogenous source of energy is not required for these reactions. Using DNA substrates containing radioactive phosphate groups, we demonstrate that IN becomes covalently joined to the new 5' phosphate ends of DNA produced at sites of cleavage. Most of the phosphodiester linkages between IN and DNA involve serine, but some involve threonine. Computer-assisted alignment of 80 retroviral and retrotransposon IN sequences identified one serine that is conserved in all of these proteins and three less-conserved threonine residues. These results identify candidate active-site residues and provide support for the participation of a covalent IN-DNA intermediate in retroviral integration.
Targeted inactivation of the insulin receptor gene in mouse 3T3-L1 fibroblasts via homologous recombination.
Authors: Accili D , Taylor S I
Published: May 2006

To study the role of the insulin receptor in determining adipocyte differentiation of the mouse cell line 3T3-L1, we have introduced a mutation that inactivates the insulin receptor gene by homologous recombination. In two independent clones, inactivation of one allele of the insulin receptor gene was associated with a 50-70% reduction in the number of insulin receptors. In addition, both clones were markedly impaired in their ability to differentiate into adipocytes. The defect in adipocyte-specific differentiation was corrected by expression of transfected human insulin receptor cDNA. These data suggest that the insulin receptor may play an important role in promoting differentiation of 3T3-L1 cells into adipocytes in vitro.
Isolation of TFC1, a gene encoding one of two DNA-binding subunits of yeast transcription factor tau (TFIIIC).
Authors: Swanson R N , Conesa C , Lefebvre O , Carles C , Ruet A , Quemeneur E , Gagnon J , Sentenac A
Published: May 2006

Transcription factor TFIIIC mediates tRNA and 5S RNA gene activation by binding to intragenic promoter elements. The factor from Saccharomyces cerevisiae, also called tau, is a large, multisubunit protein (550-650 kDa) containing two polypeptides that interact directly with DNA encoding tRNA (tDNA). We have obtained peptide sequences from the 95-kDa DNA-binding subunit (tau 95) and cloned the corresponding gene, called TFC1. The gene encodes a polypeptide of calculated Mr 73,500. However, when TFC1 was transcribed and translated in vitro, the gene product comigrated with tau 95 in SDS/polyacrylamide gels. A fusion protein expressed in bacteria was able to prevent the binding of anti-tau 95 antibodies to tau-tDNA complexes. The TFC1 gene is present in single copy on yeast chromosome II and is essential for growth. Spores containing a disrupted gene germinate but only proceed through a few cell divisions before ceasing to grow. The TFC1-encoded protein contains a potential helix-turn-helix structure and an acidic carboxyl-terminal domain, a feature characteristic of some DNA-binding proteins and transcriptional regulators.
Evidence for retrotransposition of the I factor, a LINE element of Drosophila melanogaster.
Authors: Pélisson A , Finnegan D J , Bucheton A
Published: May 2006

LINEs are transposable elements found in various eukaryotes such as plants, protists, insects, and mammals. Their transposition is usually difficult to study, particularly in humans, where some diseases have been shown to result from LINE insertion mutations. This is due to the fact that most copies of any particular family of elements are defective and that their transposition frequency is low. By contrast, the I factor of Drosophila melanogaster transposes at high frequency during I-R hybrid dysgenesis and is a good model for studying the LINE element superfamily. LINEs encode putative polypeptides showing similarities with viral reverse transcriptases but, unlike viral retrotransposons, they do not have terminal repeats and their ability to transpose by reverse transcription has previously only been inferred from structural analysis. Here we present direct evidence for LINE retrotransposition. Transposition of an I factor marked by an intron resulted in accurate removal of the intron.
Expression of type I and type II bovine scavenger receptors in Chinese hamster ovary cells: lipid droplet accumulation and nonreciprocal cross competition by acetylated and oxidized low density lipoprotein.
Authors: Freeman M , Ekkel Y , Rohrer L , Penman M , Freedman N J , Chisolm G M , Krieger M
Published: May 2006

Type I and type II scavenger receptors, which have been implicated in the development of atherosclerosis and other macrophage-associated functions, differ only by the presence in the type I receptor of an extracellular cysteine-rich C-terminal domain. Stable Chinese hamster ovary (CHO) cell transfectants expressing high levels of either the type I or type II bovine scavenger receptors have been generated. Type I and type II receptors in these cells mediated high-affinity saturable endocytosis of both 125I-labeled acetylated low density lipoprotein (LDL) and 125I-labeled oxidized LDL with the distinctive broad ligand specificity characteristic of scavenger receptors. After incubation for 2 days with acetylated LDL, the transfected cells accumulated oil red O-staining lipid droplets reminiscent of those in macrophage foam cells, whereas untransfected CHO cells did not. Thus, macrophage-specific gene products other than the scavenger receptor are not required for modified-LDL-induced intracellular lipid accumulation. In transfected cells, acetylated LDL efficiently competed for both its own endocytosis and that of oxidized LDL. In contrast, oxidized LDL competed effectively for its own endocytosis but only poorly for that of acetylated LDL. This nonreciprocal cross competition suggests that these ligands may bind to nonidentical but interacting sites on a single receptor. Results were similar for transfectants expressing either type I or type II scavenger receptors. Therefore, the nonreciprocal cross competition previously reported for cultured peritoneal macrophages may not be the result of differences between the type I and type II receptors. The nonreciprocal cross competition seen in the transfected CHO cells differs from that previously observed with cultured macrophages.
Regulation of movement speed by intracellular pH during Dictyostelium discoideum chemotaxis.
Authors: Van Duijn B , Inouye K
Published: May 2006

Evidence is presented that the chemoattractant-induced cytoplasmic alkalinization in the cellular slime mold Dictyostelium discoideum is essential in regulating locomotion speed during chemotaxis. Intracellular pH was manipulated with weak bases, weak acids, and proton-pump inhibition. Speed of locomotion of individual cells was measured during random and chemotactic movement. We found that (i) an increase of cytoplasmic pH increases the speed of randomly moving cells and (ii) the chemoattractant-induced rise in intracellular pH is essential for the increase in directed locomotion speed upon chemotactic stimulation. In addition, our experiments support the hypothesis that ammonia plays a key role in the thermo- and phototaxis of migrating slugs by increasing the locomotion speed of individual cells through changes in intracellular pH.
Identification of nucleotide pyrophosphatase/alkaline phosphodiesterase I activity associated with the mouse plasma cell differentiation antigen PC-1.
Authors: Rebbe N F , Tong B D , Finley E M , Hickman S
Published: May 2006

The protein responsible for both nucleotide pyrophosphatase (EC 3.6.1.9) and alkaline phosphodiesterase I (EC 3.1.4.1) activities was purified from MOPC 315 plasmacytoma cells. A single SDS/PAGE-purified 115-kDa protein band was used to produce a rabbit polyclonal antiserum. This antibody preparation precipitated alkaline phosphodiesterase I activity, indicating that the SDS/PAGE-purified protein was nucleotide pyrophosphatase/alkaline phosphodiesterase I. When used for Western blot analysis, the antiserum detected a 115-kDa protein as well as a 220-kDa protein band. Multiple overlapping cDNA clones were isolated from a cDNA expression library screened with this anti-nucleotide pyrophosphatase/alkaline phosphodiesterase I antiserum. Sequence analysis indicated that the isolated cDNA clones encoded PC-1, a murine plasma cell differentiation antigen. To confirm the suspected enzymatic identity of PC-1, a recombinant PC-1 fusion protein was expressed in bacteria, purified, and used to produce another rabbit polyclonal antiserum. This antiserum likewise immunoprecipitated alkaline phosphodiesterase I activity and recognized the 115-kDa and 220-kDa proteins in Western blot analyses of cell extracts. Furthermore, expression of nucleotide pyrophosphatase/alkaline phosphodiesterase I corresponded directly with mRNA and protein levels of PC-1 in cells known to express different levels of nucleotide pyrophosphatase/alkaline phosphodiesterase I activity. Finally, steroid induction of enzymatic activity was mirrored by levels of PC-1 mRNA and protein expression. Together, these data indicate that the plasma cell differentiation antigen PC-1 is a membrane-bound enzyme, nucleotide pyrophosphatase/alkaline phosphodiesterase I.
The retinoblastoma gene functions as a growth and tumor suppressor in human bladder carcinoma cells.
Authors: Takahashi R , Hashimoto T , Xu H J , Hu S X , Matsui T , Miki T , Bigo-Marshall H , Aaronson S A , Benedict W F
Published: May 2006

The product of the human retinoblastoma gene (RB) is a nuclear phosphoprotein that is thought to function as a tumor suppressor. Mutations of RB frequently occur in human bladder carcinoma. To investigate the significance of the functional loss of this gene in bladder cancer, an RB expression plasmid (pBARB) under control of the human beta-actin promoter was transfected into the bladder carcinoma cell line HTB9, which lacks RB expression. Marker-selected transfectants that expressed RB protein were identified by immunoblotting and immunohistochemical staining. In selected clones, stable RB expression has persisted over 1 yr under standard culture conditions with 10% serum. However, RB expression caused major alterations of HTB9 growth properties both in vitro and in vivo. RB+ transfectants lacked the ability to form colonies in semi-solid medium, and their growth rate was significantly decreased in 3% serum. In addition, the tumorigenicity of these transfectants was markedly decreased. Tumors that formed in nude mice were much smaller and had a longer latency period but were indistinguishable microscopically from those produced by parental cells. Slower growing tumors were RB+, as measured by nuclear staining of their RB protein and by a normal RB protein pattern on immunoblots. These findings support the concept that the RB gene acts as both a growth and tumor suppressor in bladder cancer cells.
Alpha-helix stabilization by natural and unnatural amino acids with alkyl side chains.
Authors: Lyu P C , Sherman J C , Chen A , Kallenbach N R
Published: May 2006

Knowledge of the role of individual side chains in forming different secondary structures such as the alpha-helix would be useful for prediction of protein structure from sequence or de novo protein design. Experimental and theoretical studies on natural and synthetic peptides and proteins indicate that individual side chains differ in their helix-forming potential. Four aliphatic side chains occur in the standard complement of amino acids: alanine and leucine are helix stabilizing, whereas isoleucine and valine are weakly destabilizing. We have synthesized a series of helical peptides containing unnatural aliphatic side chains having two to four carbons to explore some of the factors involved in alpha-helix stabilization and the basis for selection of the natural set. We find that linear side chains with two, three, or four carbons are as strongly helix stabilizing as the single methyl in alanine and that all linear side chains are stronger helix promoters than leucine. In addition, a t-butyl side chain is significantly more helix destabilizing than the sec-butyl side chain of isoleucine, the isopropyl side chain of valine, or even the unrestricted side chain of glycine. These results provide experimental evidence that restriction in conformational freedom of a side chain imposed by alpha-helix formation is a major component of the role of a side chain in stabilizing helical structure.
Molecular cloning of a rat testis form of the inhibitor protein of cAMP-dependent protein kinase.
Authors: Van Patten S M , Ng D C , Th'ng J P , Angelos K L , Smith A J , Walsh D A
Published: May 2006

The form of inhibitor protein of the cAMP-dependent protein kinase (PKI) that has been most thoroughly studied is a protein purified from rabbit skeletal muscle. Beale et al. previously isolated a species of PKI from rat testis that appeared from its amino acid composition to be quite distinct from the rabbit skeletal muscle protein [Beale, E. G., Dedman, J. R. & Means, A. R. (1977) J. Biol. Chem. 252, 6322-6327]. The amino acid sequence of a form of rat testis PKI has now been determined both by sequencing overlapping peptide fragments for 95% of the protein and by the isolation of a cDNA clone containing the coding region for the 70-amino acid protein. The sequence of the 70-amino acid testis PKI displays a maximum of only 41% sequence identity with the previously sequenced 75-amino acid rabbit skeletal muscle PKI. However, the two forms have identical potency as inhibitors and the key amino acids of the pseudosubstrate site, shown to be critical for maximal inhibition with the rabbit skeletal muscle PKI, have been conserved in the testis protein. The rabbit skeletal muscle and rat testis PKIs most likely represent distinct isoforms. The nucleotide sequence of the rat testis PKI cDNA suggests that a second form of testis PKI, longer by 8 additional amino-terminal amino acids, might also be produced.
Sulfate contributes to the negative charge of podocalyxin, the major sialoglycoprotein of the glomerular filtration slits.
Authors: Dekan G , Gabel C , Farquhar M G
Published: May 2006

Podocalyxin is the major sialoprotein of the rat glomerulus. Its function is to maintain the filtration slits of the glomerular epithelium open by virtue of its high net negative charge. We have used biosynthetic labeling and oligosaccharide analysis to characterize the anionic-charge-carrying moieties on this protein. Kidney slices from 2-day-old rats were biosynthetically labeled with [35S]Cys, [3H]Man, [3H]GlcN, and 35SO4, after which podocalyxin was immunoprecipitated and purified by SDS/PAGE. All these labels were incorporated into podocalyxin. Immunoprecipitates were subjected to digestion with specific glycosidases or digested with Pronase followed by chromatographic analysis of the released glycopeptides. Podocalyxin was susceptible to digestion with N-Glycanase and O-Glycanase, indicating the presence of both N- and O-linked oligosaccharides. Approximately 30% of the [3H]GlcN-labeled glycopeptides bound to Con A, confirming the presence of high mannose, hybrid, or biantennary N-linked structures; alkaline borohydride treatment confirmed the presence of O-linked oligosaccharides. Analysis of the 35SO4-labeled glycopeptides indicated that both the N- and O-linked structures were sulfated. We conclude that in newborn rat kidney (i) podocalyxin contains both O- and N-linked oligosaccharides [high mannose or hybrid type, biantennary, and complex (sialylated) type], (ii) podocalyxin is sulfated, and (iii) sulfate is located on both O-linked oligosaccharides and on glycopeptides carrying tri- or tetrantennary N-linked structures. These results indicate that the net negative charge of podocalyxin is most likely derived from sulfate as well as from sialic acid residues.
acj6: a gene affecting olfactory physiology and behavior in Drosophila.
Authors: Ayer R K , Carlson J
Published: May 2006

Mutations affecting olfactory behavior provide material for use in molecular studies of olfaction in Drosophila melanogaster. Using the electroantennogram (EAG), a measure of antennal physiology, we have found an adult antennal defect in the olfactory behavioral mutant abnormal chemosensory jump 6 (acj6). The acj6 EAG defect was mapped to a single locus and the same mutation was found to be responsible for both reduction in EAG amplitude and diminished behavioral response, as if reduced antennal responsiveness to odorant is responsible for abnormal chemosensory behavior in the mutant. acj6 larval olfactory behavior is also abnormal; the mutation seems to alter cellular processes necessary for olfaction at both developmental stages. The acj6 mutation exhibits specificity in that visual system function appears normal in larvae and adults. These experiments provide evidence that the acj6 gene encodes a product required for olfactory signal transduction.
Potent cytodifferentiating agents related to hexamethylenebisacetamide.
Authors: Breslow R , Jursic B , Yan Z F , Friedman E , Leng L , Ngo L , Rifkind R A , Marks P A
Published: May 2006

Bishydroxamic acids are effective inducers of differentiation in murine erythroleukemia cells. Flexible analogs of suberic acid bisdimethylamide are approximately 100 times as active as the parent compound or hexamethylenebisacetamide. They also induce differentiation of human promyelocytic leukemia cells (HL-60) and a subclone of human colon carcinoma cells (HT-29-U4). Some rigid bishydroxamic acids with benzene rings in the spacers are even more active toward murine erythroleukemia cells but show curious biological differences. In contrast to the flexible molecules, those with benzene spacers show poor activity toward HL-60 cells; they also have different geometric requirements, and they are not additive with hexamethylenebisacetamide in their effect. It is likely that rigid bishydroxamic acids, with a benzene ring spacer, induce differentiation by a different mechanism in spite of their chemical resemblance to the flexible bisamide and bishydroxamic acid inducers.
Induction of antibodies to the envelope protein of the human immunodeficiency virus by immunization with monoclonal anti-idiotypes.
Authors: Zaghouani H , Goldstein D , Shah H , Anderson S , Lacroix M , Dionne G , Kennedy R , Bona C
Published: May 2006

Anti-idiotypes that possess the internal image of antigen can induce protective humoral immunity toward microbes. Herein we demonstrate antigen mimicry by monoclonal anti-idiotypes of a distinct epitope of the human immunodeficiency virus (HIV) envelope protein that is defined by a synthetic peptide. This peptide, corresponding to amino acid residues 503-535 (peptide 503-535) of HIV-1 IIIB gp160, induced antibodies in three mammalian species that interacted with HIV-1 gp120 and inhibited in vitro syncytium formation caused by HIV-1, IIIB and MN isolates. Three monoclonal anti-idiotypes were generated against rabbit anti-gp120 antibodies specific for peptide 503-535. These anti-idiotypes recognize an interspecies cross-reactive idiotype expressed on mouse, chimpanzee, baboon, rabbit, and human anti-gp120 antibodies specific for peptide 503-535. The interaction with the cross-reactive idiotype is inhibited by synthetic peptide and HIV-1 gp160. Furthermore, rabbits immunized with the monoclonal anti-idiotypes produced antibodies that also bind HIV-1 gp120 and gp160 and recognized the epitope defined by peptide 503-535.
Evidence for regulation of the human ABL tyrosine kinase by a cellular inhibitor.
Authors: Pendergast A M , Muller A J , Havlik M H , Clark R , McCormick F , Witte O N
Published: May 2006

Phosphotyrosine cannot be detected on normal human ABL protein-tyrosine kinases, but activated oncogenic forms of the human ABL protein are phosphorylated on tyrosine in vivo. Activation of ABL can occur by substitution of the ABL first exon with breakpoint cluster region (BCR) sequences or by deletion of the noncatalytic SH3 (src homology region 3) domain. An alternative mode for the activation of the ABL kinases is hyperexpression at greater than 500-fold over endogenous levels. This is not a consequence of transphosphorylation of the hyperexpressed ABL molecules. ABL proteins translated in vitro lack phosphotyrosine, but tyrosine kinase activity is uncovered after immunoprecipitation and removal of lysate components. The rates of dephosphorylation of ABL and BCR-ABL fusion protein by phosphotyrosine-specific phosphatases are approximately the same. These combined results indicate that inhibition of ABL activity is reversible and suggest that a cellular component interacts noncovalently with ABL to inhibit its autophosphorylation.
A donor splice mutation and a single-base deletion produce two carboxyl-terminal variants of human serum albumin.
Authors: Watkins S , Madison J , Davis E , Sakamoto Y , Galliano M , Minchiotti L , Putnam F W
Published: May 2006

At least 35 allelic variants of human serum albumin have been sequenced at the protein level. All except two COOH-terminal variants, Catania and Venezia, are readily explainable as single-point substitutions. The two chain-termination variants are clustered in certain locations in Italy and are found in numerous unrelated individuals. In order to correlate the protein change in these variants with the corresponding DNA mutation, the two variant albumin genes have been cloned, sequenced, and compared to normal albumin genomic DNA. In the Catania variant, a single base deletion and subsequent frameshift leads to a shortened and altered COOH terminus. Albumin Venezia is caused by a mutation that alters the first consensus nucleotide of the 5' donor splice junction of intron 14 and the 3' end of exon 14, which is shortened from 68 to 43 base pairs. This change leads to an exon skipping event resulting in direct splicing of exon 13 to exon 15. The predicted Venezia albumin product has a truncated amino acid sequence (580 residues instead of 585), and the COOH-terminal sequence is altered after Glu-571. The variant COOH terminus ends with the dibasic sequence Arg-Lys that is apparently removed through stepwise cleavage by serum carboxypeptidase B to yield several forms of circulating albumin.
Molecular cloning and characterization of the yeast gene for squalene synthetase.
Authors: Jennings S M , Tsay Y H , Fisch T M , Robinson G W
Published: May 2006

Squalene synthetase (farnesyl-diphosphate: farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21) is a critical branch point enzyme of isoprenoid biosynthesis that is thought to regulate the flux of isoprene intermediates through the sterol pathway. The structural gene for this enzyme was cloned from the yeast Saccharomyces cerevisiae by functional complementation of a squalene synthetase-deficient erg9 mutant. Identification of this ERG9 clone was confirmed by genetic linkage analysis in yeast and expression of enzyme activity in Escherichia coli. The predicted squalene synthetase polypeptide of 444 amino acids (Mr, 51,753) lacks significant homology to known protein sequences, except within a region that may represent a prenyl diphosphate (substrate) binding site. The ERG9-encoded protein contains a PEST consensus motif (rich in proline, glutamic acid, serine, and threonine) present in many proteins with short cellular half-lives. Modeling of the protein suggests that it contains at least one, and possibly two, membrane-spanning domains. Disruption of the chromosomal squalene synthetase coding region by insertional mutagenesis indicates that ERG9 is a single copy gene that is essential for cell growth in yeast.
Human chromosome 16 encodes a factor involved in induction of class II major histocompatibility antigens by interferon gamma.
Authors: Bono M R , Alcaïde-Loridan C , Couillin P , Letouzé B , Grisard M C , Jouin H , Fellous M
Published: May 2006

Interferon gamma (IFN-gamma) induces expression of class II major histocompatibility complex (MHC)-encoded antigens in immunocompetent cells. To gain further insight into the mechanism of this induction, we prepared somatic cell hybrids between different human cell lines and a murine cell line, RAG, that does not express murine class II MHC antigens before or after treatment with murine IFN-gamma. Some of the resulting cell hybrids express murine class II MHC antigens when treated with murine IFN-gamma. This inducible phenotype is correlated with the presence of human chromosome 16. It has been shown previously that the induction of class I MHC antigens by human IFN-gamma in human-rodent hybrids requires the presence of species-specific factors encoded by chromosome 6, which bears the gene for the human IFN-gamma receptor, and chromosome 21, whose product(s) is necessary for the transduction of human IFN-gamma signals. In this report, we show that the induction of murine class II MHC antigens by human IFN-gamma in the human-RAG cell hybrids requires, likewise, the presence of human chromosomes 6 and 21, in addition to chromosome 16. In some of these hybrids, when all three of these human chromosomes were present, induction of cell-surface HLA-DR antigens was also observed. Our results demonstrate that human chromosome 16 encodes a non-species-specific factor involved in the induction of class II MHC antigens by IFN-gamma.
A P element of Scaptomyza pallida is active in Drosophila melanogaster.
Authors: Simonelig M , Anxolabéhère D
Published: May 2006

Several results suggest that P elements have recently invaded natural populations of Drosophila melanogaster after a horizontal transfer from another species. The donor species is thought to come from the willistoni group, which contains P elements very homologous to those of D. melanogaster. However, more divergent P elements are present in many other Drosophilidae species. We have analyzed such elements from Scaptomyza pallida, a species phylogenetically distant to D. melanogaster. We report here the isolation of two coding P elements from S. pallida (PS2 and PS18) that are 4% divergent from one another. At least one of these elements (PS18) is active since it is able to transpose in D. melanogaster and to mobilize a D. melanogaster defective P element, even though its nucleotide sequence is 24% divergent from the canonical P element of D. melanogaster. To our knowledge, a P element that is active and strongly divergent from the D. melanogaster P element has not been reported previously. Sequence comparison between the complete P elements of D. melanogaster and S. pallida reveals that the structural characteristics are maintained: PS2 and PS18 contain terminal inverted repeats and internal repeats very similar to those of the D. melanogaster P element. In addition, the noncoding regions cis necessary for the transposition are more conserved than the coding sequences. Two domains found in the D. melanogaster P transposase (helix-turn-helix and leucine zipper) are well conserved in the putative proteins encoded by PS2 and PS18. This study provides insights into which parts of P elements are functionally important and correlates with functional studies of the P element in D. melanogaster.
Anionic subsites of the catalytic center of acetylcholinesterase from Torpedo and from cobra venom.
Authors: Kreienkamp H J , Weise C , Raba R , Aaviksaar A , Hucho F
Published: May 2006

A peptide of acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7) from the venom of the cobra Naja naja oxiana labeled by the affinity reagent N,N-dimethyl-2-phenylaziridinium (DPA) has been identified. The sequence is Gly-Ala-Glu-Met-Trp-Asn-Pro-Asn. In AcChoEase from Torpedo californica, a homologous peptide was labeled and isolated. Its sequence is Ser-Gly-Ser-Glu-Met-Trp-Asn-Pro-Asn, representing positions 79 through 87. In both cases labeling can be prevented by 0.1 mM edrophonium, indicating that the respective peptides form part of the anionic subsite of the catalytic center. The modified residue was tryptophan (Trp-84 in Torpedo AcChoEase) in both enzymes. In contrast to AcChoEase from Torpedo, the enzyme from cobra venom does not contain a peripheral anionic binding site.
Interaction of Escherichia coli tRNA(Ser) with its cognate aminoacyl-tRNA synthetase as determined by footprinting with phosphorothioate-containing tRNA transcripts.
Authors: Schatz D , Leberman R , Eckstein F
Published: May 2006

A footprinting technique using phosphorothioate-containing RNA transcripts has been developed and applied to identify contacts between Escherichia coli tRNA(Ser) and its cognate aminoacyl-tRNA synthetase. The cloned gene for the tRNA was transcribed in four reactions in which a different NTP was complemented by 5% of the corresponding nucleoside 5'-O-(1-thiotriphosphate). The phosphorothioate groups of such transcripts are cleaved by reaction with iodine to permit sequencing of the transcripts. Footprinting was achieved by performing the same reaction with the phosphorothioate-tRNA-enzyme complex. At 1 mM iodine, selective protection of the tRNA transcripts in the cognate system was observed, with strong protection at positions 52 and 68 and weak protection at positions 46, 53, 67, 69, and 70. It is suggested that these regions of the tRNA interact with the helical arm of the synthetase.
Post-translational modification of microtubules is a component of synergic alterations of cytoskeleton leading to formation of cytoplasmic processes in fibroblasts.
Authors: Tint I S , Bershadsky A D , Gelfand I M , Vasiliev J M
Published: May 2006

The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) induces rapid and reversible shape changes in cultured fibroblasts: extension of motile lamellas is followed by transformation of these lamellas into nonmotile stalklike processes. This "lamella-to-stalk" transformation was found to be associated with the formation of microtubules enriched in detyrosinated alpha-tubulin. This change was local: microtubules in motile lamellas at the distal ends of the processes and in the cell bodies were not enriched in detyrosinated alpha-tubulin. Detyrosinated microtubules in the processes were more resistant to Colcemid treatment than other microtubules of PMA-treated and control cells. The effects of PMA were reversible and could be abolished by sphingosine, a specific inhibitor of protein kinase C. Besides modification of microtubules, lamella-to-stalk transformation is associated with the ingrowth of intermediate filaments into the extensions. Earlier it was found that this transformation is also associated with the profound reorganization of the system of actin microfilaments. Thus, all three cytoskeletal systems are altered simultaneously during PMA-induced formation of processes. Similar "cytoskeletal synergies" may play essential roles in many morphogenetic processes--e.g., in the growth of neurites.
Cytotactin expression in somites after dorsal neural tube and neural crest ablation in chicken embryos.
Authors: Tan S S , Prieto A L , Newgreen D F , Crossin K L , Edelman G M
Published: May 2006

The spatiotemporal expression of the extracellular matrix protein cytotactin/tenascin during somitogenesis suggests that it plays a role in the morphogenetic events that give rise to the pattern of neural crest (NC) development. In the present study, the spatial distribution and molecular forms of cytotactin in somites were examined using in situ hybridization, Western blotting, and immunohistochemistry during normal development and after injury. In situ hybridization showed that prior to NC cell invasion cytotactin mRNA was restricted to the caudal half of the newly formed epithelial somites. As each epithelial somite matured, giving rise to a sclerotome and dermamyotome, the mRNA was first restricted to the dermamyotome and later restricted to the rostral protion of the sclerotome, consistent with the previously reported protein distribution. Immunocytochemical analysis of the distribution of cytotactin and NC cells in embryos with ablations that removed NC cells, or with simple wounds that left NC cells in place, demonstrated that the presence of NC cells is neither necessary nor sufficient for the correct positioning of cytotactin. Immunoblotting analysis showed that cytotactin synthesized by sclerotomes in the absence of NC cells was of similar molecular mass to that produced in their presence. These findings are in accord with the notion that the abnormalities of cytotactin distribution are related to the wounding process. We conclude that, contrary to the suggestion of Stern et al. [Stern, C. D., Norris, W. E., Bronner-Fraser, M., Carlson, G. J., Faissner, A., Keynes, R. J. & Schachner, M. (1989) Development 107, 309-319], there is no causal link between the presence of NC cells and the distribution and molecular mass of sclerotomal cytotactin.