bluuebunny/crossref_metadata_2025 huggingface dataset:
Permeability of the murine blood-brain barrier to some octapeptide analogs of somatostatin.
Authors: Banks W A , Schally A V , Barrera C M , Fasold M B , Durham D A , Csernus V J , Groot K , Kastin A J
Published: May 2006

Analogs of somatostatin are being investigated clinically for the treatment of various malignancies, including brain tumors. We studied the ability of three therapeutically promising radioactively labeled somatostatin octapeptide analogs, RC-160, RC-121, and RC-161, to cross the blood-brain barrier (BBB) after peripheral or central injection. After i.v. injection, intact RC-160 was recovered from the blood and the brain. The entry rates were different from each compound but were generally low. By contrast, entry across the intact BBB increased 220 times when RC-160 was given in a serum-free perfusate. This suggests that some serum-related factor, probably the previously described protein binding or an aggregation-promoting factor, is the main determinant in limiting the blood-to-brain passage of somatostatin analogs. Entry into the brain was not inhibited by the addition of unlabeled analog to the perfusate, showing that passage was probably by diffusion across the membranes that comprise the BBB rather than by saturable transport. By contrast, a saturable system was found to transport peptide out of the central nervous system (CNS). The clearance from the CNS of RC-160 and RC-121, but not RC-161, was faster than could be accounted for by reabsorption of cerebrospinal fluid. Transport of radioactively labeled RC-160 out of the CNS was inhibited by unlabeled RC-160 or somatostatin but was not affected by some other peptide known to cross the BBB by their own transport systems. More than 80% of the radioactivity recovered from the blood after intracerebroventricular injection of RC-160 was eluted by HPLC at the position of the labeled analog, showing that the peptide had crossed the BBB in intact form. Our results indicate the presence of a saturable transport system in one direction across the BBB for some superactive analogs of somatostatin.
Allele loss on chromosome 16 associated with progression of human hepatocellular carcinoma.
Authors: Tsuda H , Zhang W D , Shimosato Y , Yokota J , Terada M , Sugimura T , Miyamura T , Hirohashi S
Published: May 2006

Loss of heterozygosity on chromosome 16 is a common genetic alteration in human hepatocellular carcinoma (HCC). To clarify the pathogenetic significance of allele loss on chromosome 16, we performed restriction fragment length polymorphism analysis of 70 surgically resected tumors by using 15 polymorphic DNA markers for chromosome 16. Loss of heterozygosity on chromosome 16 was detected in 36 (52%) of 69 informative cases, and the common region of allele loss in these 36 tumors was located between the HP locus (16q22.1) and the CTRB locus (16q22.3-q23.2). These losses occurred more frequently in HCCs of poor differentiation, of larger size, and with metastasis, whereas they were not detected in HCC at the earliest stage. In addition, these losses were not associated with presence or absence of hepatitis B virus DNA integration or hepatitis C virus infection. These results show that loss of heterozygosity on chromosome 16 is a late event occurring after hepatocarcinogenesis and strongly suggest that this phenomenon is involved in enhancement of tumor aggressiveness during progression of HCC.
Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembrane antigens.
Authors: Szala S , Kasai Y , Steplewski Z , Rodeck U , Koprowski H , Linnenbach A J
Published: May 2006

The human tumor-associated antigen CO-029 is a monoclonal antibody-defined cell surface glycoprotein of 27-34 kDa. By using the high-efficiency COS cell expression system, a full-length cDNA clone for CO-029 was isolated. When transiently expressed in COS cells, the cDNA clone directed the synthesis of an antigen reactive to monoclonal antibody CO-029 in mixed hemadsorption and immunoblot assays. Sequence analysis revealed that CO-029 belongs to a family of cell surface antigens that includes the melanoma-associated antigen ME491, the leukocyte cell surface antigen CD37, and the Sm23 antigen of the parasitic helminth Schistosoma mansoni. CO-029 and ME491 antigen expression and the effect of their corresponding monoclonal antibodies on cell growth were compared in human tumor cell lines of various histologic origins.
Pathogenic anti-DNA autoantibody-inducing T helper cell lines from patients with active lupus nephritis: isolation of CD4-8- T helper cell lines that express the gamma delta T-cell antigen receptor.
Authors: Rajagopalan S , Zordan T , Tsokos G C , Datta S K
Published: May 2006

The antigen responsible for autoimmunization in systemic lupus erythematosus is unknown. In spite of this obstacle, we show that T helper (Th) cell lines that are functionally relevant to this disease can be established in vitro. We derived a total of 396 interleukin 2-dependent T-cell lines from the in vivo activated T cells of five patients with lupus nephritis. Only 59 (approximately 15%) of these lines had the ability to selectively augment the production of pathogenic anti-DNA autoantibodies that were IgG in class, cationic in charge, specific for native DNA, and clonally restricted in spectrotype. Forty-nine of these autoantibody-inducing Th lines were CD4+ and expressed the alpha beta T-cell receptor (TCR). The other 10 were CD4-8- (double negative), 3 expressing the alpha beta TCR and 7 expressing the gamma delta TCR. All of the autoantibody-inducing Th lines responded to some endogenous antigen presented by autologous B cells. The autoreactive responses of the CD4+ Th lines were restricted to HLA class II antigens, whereas those of the double-negative cells were not. Endogenous heat shock or stress proteins of the HSP60 family that were expressed by the lupus patients' B cells were involved in stimulating an autoreactive proliferation of the gamma delta Th cells. These studies demonstrate a novel helper activity of certain gamma delta T cells in a spontaneous autoimmune response.
Molecular basis of preferential resistance to colchicine in multidrug-resistant human cells conferred by Gly-185----Val-185 substitution in P-glycoprotein.
Authors: Safa A R , Stern R K , Choi K , Agresti M , Tamai I , Mehta N D , Roninson I B
Published: May 2006

Expression of P-glycoprotein, encoded by the human MDR1 gene, results in cross-resistance to many lipophilic cytotoxic drugs (multidrug resistance). P-glycoprotein is believed to function as an energy-dependent efflux pump that is responsible for decreased drug accumulation in multidrug-resistant cells. Previous work showed that preferential resistance to colchicine in a colchicine-selected multidrug-resistant cell line was caused by spontaneous mutations in the MDR1 gene that resulted in a Gly-185----Val-185 substitution in P-glycoprotein. We have now compared transfectant cell lines expressing either the wild-type Gly-185 or the mutant Val-185 P-glycoprotein with regard to their levels of resistance to and accumulation and binding of different drugs. In cells expressing the mutant protein, increased resistance to colchicine and decreased resistance to vinblastine correlated with a decreased accumulation of colchicine and increased accumulation of vinblastine. Expression of the mutant P-glycoprotein also resulted in significantly increased resistance to epipodophyllotoxin and decreased resistance to vincristine and actinomycin D; smaller changes in resistance were observed for several other drugs. Unexpectedly, the mutant P-glycoprotein showed increased binding of photoactive analogs of vinblastine and verapamil and the photoactive compound azidopine and decreased binding of a photoactive colchicine analog. These results suggest that the Gly-185----Val-185 substitution affects not the initial drug-binding site of P-glycoprotein but another site, associated with the release of P-glycoprotein-bound drugs to the outside of the cell.
Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers.
Authors: Garin-Chesa P , Old L J , Rettig W J
Published: May 2006

The F19 antigen is a cell surface glycoprotein (Mr, 95,000) of human sarcomas and proliferating, cultured fibroblasts that is absent from resting fibroblasts in normal adult tissues. Normal and malignant epithelial cells are also F19-. The present immunohistochemical study describes induction of F19 in the reactive mesenchyme of epithelial tumors. F19+ fibroblasts were found in primary and metastatic carcinomas, including colorectal (18 of 18 cases studied), breast (14/14), ovarian (21/21), bladder (9/10), and lung carcinomas (13/13). In contrast, the stroma of benign colorectal adenomas, fibrocystic disease and fibroadenomas of breast, benign prostate hyperplasia, in situ bladder carcinomas, and benign ovarian tumors showed no or only moderate numbers of F19+ fibroblasts. Analysis of dermal incision wounds revealed that F19 is strongly induced during scar formation. Comparison of F19 with the extracellular matrix protein tenascin, a putative marker of tumor mesenchyme, showed a cellular staining pattern for F19 vs. the extracellular matrix pattern for tenascin and widespread expression of tenascin in F19- normal tissues and benign tumors. Our results suggest that the F19+ phenotype correlates with specialized fibroblast functions in wound healing and malignant tumor growth. Because of its abundance in tumor mesenchyme, F19 may serve as a target for antibodies labeled with radioisotopes or toxic agents, or inflammatogenic antibodies, in carcinoma patients.
Isolation of diphtheria toxin-sensitive mouse cells from a toxin-resistant population transfected with monkey DNA.
Authors: Naglich J G , Eidels L
Published: May 2006

Diphtheria toxin (DTX)-sensitive mouse cells were isolated from a toxin-resistant thymidine kinase (TK)-negative L-M(TK-) mouse cell population that was transfected with DNA from highly toxin-sensitive monkey Vero cells. Sensitivity to DTX was screened by using a replica plate assay. The purified toxin-sensitive mouse cells were characterized with respect to their ability to bind, internalize, and translocate DTX into the cytosol. In contrast to the L-M(TK-) cells, these DTX-sensitive mouse cells were able to bind and internalize radioiodinated toxin into intracellular vesicles at 37 degrees C. Specific binding of radioiodinated toxin to their cell surface (at 4 degrees C) could not be demonstrated. However, the following evidence for functional receptors capable of binding DTX was obtained: (i) when the toxin-sensitive mouse cells were first allowed to bind DTX at 4 degrees C, followed by washing the cells and shifting the temperature to 37 degrees C (allowing cell surface-bound toxin to enter the cells), the cells were killed; (ii) when cells with surface-bound DTX were exposed briefly to an acidic medium (allowing the toxin to penetrate the plasma membrane directly), protein synthesis was inhibited; and (iii) when cells were incubated with DTX in the presence of the CRM 197, a nontoxic form of DTX with binding properties similar to native DTX, the cytotoxic effect of DTX was markedly decreased. The results demonstrate that the toxin-sensitive mouse cells are killed by a mechanism similar to that observed in naturally occurring toxin-sensitive cell lines. The data further suggest that the transfected mouse cells express functional receptors for DTX.
Direct evidence for translational regulation by leader RNA and Tat protein of human immunodeficiency virus type 1.
Authors: SenGupta D N , Berkhout B , Gatignol A , Zhou A M , Silverman R H
Published: May 2006

Translational effects of the RNA leader and Tat protein of human immunodeficiency virus type 1 (HIV-1) were investigated in rabbit reticulocyte lysate. Hybrid RNA species with natural or mutated HIV-1 leader fused to human interferon- gamma mRNA were produced in vitro from recombinant plasmids. HIV-1 leader RNA was found to inhibit translation through two mechanisms. A 3-fold trans-inhibition of translation was demonstrated by mixing hybrid HIV-1 leader RNA with indicator interferon mRNA. By comparison, HIV-1 leader caused a 50-fold cis-inhibition in lysate in which two trans-inhibitory factors, double-stranded RNA-dependent protein kinase and (2'-5')oligoadenylate synthetase, were suppressed. In contrast, purified HIV-1 Tat protein produced in Escherichia coli enhanced by 4-fold translation from HIV-1 leader-interferon mRNA but not from interferon mRNA lacking HIV sequences or from total poly(A)+ RNA. Translation of mRNA containing either a single base substitution in the loop of the "trans-acting responsive" sequence (TAR) or an alternative stem-loop in TAR was nevertheless stimulated by Tat. The enhancement of translation by Tat was largely due to relief of cis-inhibition, since the effect was found even in lysate in which double-stranded RNA-dependent protein kinase was inhibited with 2-aminopurine. These results suggest that translation is an important level of control in the replication cycle of HIV-1.
Molecular cloning of a cDNA encoding interleukin 11, a stromal cell-derived lymphopoietic and hematopoietic cytokine.
Authors: Paul S R , Bennett F , Calvetti J A , Kelleher K , Wood C R , O'Hara R M , Leary A C , Sibley B , Clark S C , Williams D A
Published: May 2006

Hematopoiesis occurs in close association with a complex network of cells loosely termed the hematopoietic microenvironment. Analysis of the mechanisms of microenvironmental regulation of hematopoiesis has been hindered by the complexity of the microenvironment as well as the heterogeneity of hematopoietic stem cells and early progenitor cells. We have established immortalized primate bone marrow-derived stromal cell lines to facilitate analysis of the interactions of hematopoietic cells with the microenvironment in a large animal species. One such line, PU-34, was found to produce a variety of growth factors, including an activity that stimulates the proliferation of an interleukin 6-dependent murine plasmacytoma cell line. A cDNA encoding the plasmacytoma stimulatory activity was isolated through functional expression cloning in mammalian cells. The nucleotide sequence contained a single long reading frame of 597 nucleotides encoding a predicted 199-amino acid polypeptide. The amino acid sequence of this cytokine, designated interleukin 11 (IL-11), did not display significant similarity with any other sequence in the GenBank data base. Preliminary biological characterization indicates that in addition to stimulating plasmacytoma proliferation, IL-11 stimulates the T-cell-dependent development of immunoglobulin-producing B cells and synergizes with IL-3 in supporting murine megakaryocyte colony formation. These properties implicate IL-11 as an additional multifunctional regulator in the hematopoietic microenvironment.
Reassociation with beta 2-microglobulin is necessary for Kb class I major histocompatibility complex binding of exogenous peptides.
Authors: Rock K L , Rothstein L E , Gamble S R , Benacerraf B
Published: May 2006

T lymphocytes recognize endogenously produced antigenic peptides in association with major histocompatibility complex (MHC)-encoded molecules. Peptides from the extracellular fluid can be displayed in association with class I and class II MHC molecules. Here we report that mature Kb class I MHC molecules bind peptides upon dissociation and reassociation of their light chain. Intact Kb heterodimers, unlike class II MHC molecules, are relatively unreceptive to binding peptides. This property may maintain segregation of class I and class II MHC-restricted peptides and has implications for the use of peptides as vaccines.
The gamma subunit of transducin is farnesylated.
Authors: Lai R K , Perez-Sala D , Cañada F J , Rando R R
Published: May 2006

Protein prenylation with farnesyl or geranylgeranyl moieties is an important posttranslational modification that affects the activity of such diverse proteins as the nuclear lamins, the yeast mating factor mata, and the ras oncogene products. In this article, we show that whole retinal cultures incorporate radioactive mevalonic acid into proteins of 23-26 kDa and one of 8 kDa. The former proteins are probably the "small" guanine nucleotide-binding regulatory proteins (G proteins) and the 8-kDa protein is the gamma subunit of the well-studied retinal heterotrimeric G protein (transducin). After deprenylating purified transducin and its subunits with Raney nickel or methyl iodide/base, the adducted prenyl group can be identified as an all-trans-farnesyl moiety covalently linked to a cysteine residue. Thus far, prenylation reactions have been found to occur at cysteine in a carboxyl-terminal consensus CAAX sequence, where C is the cysteine, A is an aliphatic amino acid, and X is undefined. Both the alpha and gamma subunits of transducin have this consensus sequence, but only the gamma subunit is prenylated. Therefore, the CAAX motif is not necessary and sufficient to direct prenylation. Finally, since transducin is the best understood G protein, both structurally and mechanistically, the discovery that it is farnesylated should allow for a quantitative understanding of this post-translational modification.
Identification of a groES-like chaperonin in mitochondria that facilitates protein folding.
Authors: Lubben T H , Gatenby A A , Donaldson G K , Lorimer G H , Viitanen P V
Published: May 2006

Mitochondria contain a polypeptide that is functionally equivalent to Escherichia coli chaperonin 10 (cpn10; also known as groES). This mitochondrial cpn10 has been identified in beef and rat liver and is able to replace bacterial cpn10 in the chaperonin-dependent reconstitution of chemically denatured ribulose-1,5-bisphosphate carboxylase. Thus, like the bacterial homologue, mitochondrial cpn10 facilitates a K(+)- and Mg.ATP-dependent discharge of unfolded (or partially folded) ribulose bisphosphate carboxylase from bacterial chaperonin 60 (cpn60; also known as groEL). Instrumental to its identification, mitochondrial cpn10 and bacterial cpn60 form a stable complex in the presence of Mg.ATP. Bacterial and mitochondrial cpn10 compete for a common saturable site on bacterial cpn60. As a result of complex formation, with either mitochondrial or bacterial cpn10, the "uncoupled ATPase" activity of bacterial cpn60 is virtually abolished. The most likely candidate for mitochondrial cpn10 is an approximately 45-kDa oligomer composed of approximately 9-kDa subunits. We propose that, like the protein-folding machinery of prokaryotes, mitochondrial cpn60 requires a cochaperonin for full biological function.
Nature's chemicals and synthetic chemicals: comparative toxicology.
Authors: Ames B N , Profet M , Gold L S
Published: May 2006

The toxicology of synthetic chemicals is compared to that of natural chemicals, which represent the vast bulk of the chemicals to which humans are exposed. It is argued that animals have a broad array of inducible general defenses to combat the changing array of toxic chemicals in plant food (nature's pesticides) and that these defenses are effective against both natural and synthetic toxins. Synthetic toxins such as dioxin are compared to natural chemicals, such as indole carbinol (in broccoli) and ethanol. Trade-offs between synthetic and natural pesticides are discussed. The finding that in high-dose tests, a high proportion of both natural and synthetic chemicals are carcinogens, mutagens, teratogens, and clastogens (30-50% for each group) undermines current regulatory efforts to protect public health from synthetic chemicals based on these tests.
Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I: [Fe-S] cluster-driven protein rearrangement.
Authors: Martín A E , Burgess B K , Stout C D , Cash V L , Dean D R , Jensen G M , Stephens P J
Published: May 2006

Azotobacter vinelandii ferredoxin I is a small protein that contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. Recently the x-ray crystal structure has been redetermined and the fdxA gene, which encodes the protein, has been cloned and sequenced. Here we report the site-directed mutation of Cys-20, which is a ligand of the [4Fe-4S] cluster in the native protein, to alanine and the characterization of the protein product by x-ray crystallographic and spectroscopic methods. The data show that the mutant protein again contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. The new [4Fe-4S] cluster obtains its fourth ligand from Cys-24, a free cysteine in the native structure. The formation of this [4Fe-4S] cluster drives rearrangement of the protein structure.
A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells.
Authors: Jerse A E , Yu J , Tall B D , Kaper J B
Published: May 2006

The ability of enteropathogenic Escherichia coli (EPEC) to form attaching and effacing intestinal lesions is a major characteristic of EPEC pathogenesis. Using TnphoA mutagenesis we have identified a chromosomal gene (eae, for E. coli attaching and effacing) that is necessary for this activity. A DNA probe derived from this gene hybridizes to 100% of E. coli of EPEC serogroups that demonstrate attaching and effacing activity on tissue culture cells as well as other pathogenic E. coli that produce attaching and effacing intestinal lesions, such as RDEC-1 (an EPEC of weanling rabbits) and enterohemorrhagic E. coli. The predicted amino acid sequence derived from the nucleotide sequence of eae shows significant homology to that of the invasin of Yersinia pseudotuberculosis.
Parallel zippers formed by alpha-helical peptide columns in crystals of Boc-Aib-Glu(OBzl)-Leu-Aib-Ala-Leu-Aib-Ala-Lys(Z)-Aib-OMe.
Authors: Karle I L , Flippen-Anderson J L , Uma K , Balaram P
Published: May 2006

The crystal structure of the decapeptide Boc-Aib-Glu(OBzl)-Leu-Aib-Ala-Leu-Aib-Ala-Lys(Z)-Aib-OMe (where Aib is alpha-aminoisobutyryl, Boc is t-butoxycarbonyl, OBzl is benzyl ester, and Z is benzyloxycarbonyl) illustrates a parallel zipper arrangement of interacting helical peptide columns. Head-to-tail NH...OC hydrogen bonding extends the alpha-helices formed by the decapeptide into long columns in the crystal. An additional NH...OC hydrogen bond in the head-to-tail region, between the extended side chains of Glu(OBzl), residue 2 in one molecule, and Lys(Z), residue 9 in another molecule, forms a "double tooth" on the side of the column. These double teeth are repeated regularly on the helical columns with spaces of six residues between them (approximately 10 A). The double teeth on a pair of parallel columns (all carbonyl groups pointed in the same direction) interdigitate in a zipper motif. All contacts in the zipper portion are of the van der Waals type. The peptide, with formula C66H103N11O17.H2O, crystallizes in space group P2(1)2(1)2(1) with a = 10.677(4) A, b = 16.452(6) A, and c = 43.779(13) A; overall agreement R = 10.2% for 3527 observed reflections (magnitude of /F0/ greater than 3 sigma); resolution 0.9 A.
Location of the cis-acting auxin-responsive region in the promoter of the par gene from tobacco mesophyll protoplasts.
Authors: Takahashi Y , Niwa Y , Machida Y , Nagata T
Published: May 2006

We have isolated a genomic clone of an auxin-regulated par gene, which is expressed during the transition from G0 phase to S phase in the early stage of tobacco mesophyll protoplasts cultured in vitro, from a tobacco genomic library using the par cDNA as a probe. When a chimeric gene, in which a reporter gene for bacterial beta-glucuronidase (GUS) was placed downstream of the 5' flanking sequences of the par gene, was introduced into tobacco mesophyll protoplasts by electroporation, the chimeric gene elicited auxin-regulated expression of GUS activity. Because deletion of a 111-base-pair (bp) direct repeat in the 5' flanking sequences of the par gene abolished the auxin-induced GUS activity, it is deduced that in the 111-bp direct repeat of the par gene promoter is localized an auxin-responsive region, which regulates auxin-mediated activation of transcription.
Chimeric phage-bacterial enzymes: a clue to the modular evolution of genes.
Authors: Díaz E , López R , García J L
Published: May 2006

Pneumococcal peptidoglycan amidase (N-acetylmuramoyl-L-alanine amidase, EC 3.5.1.28) and phage CPL1 lysozyme degrade a common substrate (choline-containing pneumococcal cell walls); the former hydrolyzes the bond between muramic acid and alanine, whereas the latter breaks down the linkage between muramic acid and glucosamine. The amino acid sequences of their C-terminal domains are homologous. Chimeric genes were constructed by site-directed mutagenesis: a unique SnaBI restriction site in the cpl1 gene, coding for the phage lysozyme, was introduced at a location equivalent to the SnaBI site present in the lytA gene, which codes for the pneumococcal amidase. The resulting genes expressed lytic activities at levels similar to those of the parental genes. The gene products, which have been purified to electrophoretical homogeneity, exhibited unusual combined biochemical properties--e.g., by exchange of protein domains, we have switched the regulatory properties of these enzymes without altering their catalytic activities. Chimeric gene construction in Streptococcus pneumoniae and its bacteriophages is an excellent model to study the modular organization of genes and proteins and to help to establish evolutionary relationships between phage and bacteria. These constructions provide an experimental approach to the molecular processes involved in cassette recruitment during evolution and contribute support to the concept of bacteria as adaptable chimeras.
Thyrotropin-releasing-hormone (TRH) and its physiological metabolite TRH-OH inhibit Na+ channel activity in mammalian septal neurons.
Authors: López-Barneo J , Castellano A , Toledo-Aral J
Published: May 2006

The interaction of thyrotropin-releasing hormone (TRH) and its physiological metabolite TRH-OH with Na+ channels was studied in enzymatically dissociated guinea pig septal neurons by using the whole-cell variant of the patch-clamp technique. In about 60% of the cells tested, the neuropeptides at concentrations between 0.01 and 2.5 microM produced a dose-dependent reversible attenuation of Na+ currents. With 2 microM TRH-OH, peak Na+ current amplitude was reduced by 20-50% (27 +/- 8%, mean +/- SD; n = 16), whereas at the same concentration TRH was approximately half as effective as TRH-OH. In the presence of the tripeptides, the voltage-dependent parameter of the Na+ current were unaltered. TRH-induced reduction of Na+ current amplitude was transient and recovered almost completely during maintained exposure to the peptides. In addition, the response to either TRH-OH or TRH decreased with repeated treatment. Our results demonstrate that neuronal Na+ channels can be modulated by naturally occurring neuropeptides.
Overexpression of eukaryotic protein synthesis initiation factor 4E in HeLa cells results in aberrant growth and morphology.
Authors: De Benedetti A , Rhoads R E
Published: May 2006

Eukaryotic protein synthesis initiation factor 4E (eIF-4E) is a 25-kDa polypeptide that binds to the 7-methylguanosine-containing cap of mRNA and participates in the transfer of mRNA to the 40S ribosomal subunit, a step that is rate-limiting for protein synthesis under most cellular conditions. eIF-4E is the least abundant of the initiation factors, is present at approximately 10% of molar concentration of mRNA, and thus may serve as a site of regulation for the recruitment of mRNA into polysomes. Previous studies have indicated that phosphorylation of eIF-4E at Ser-53 is correlated with an increased rate of protein synthesis in a variety of systems in vivo and is required for eIF-4E to become bound to the 48S initiation complex. In this study we show that overexpression of eIF-4E in HeLa cells using an episomally replicating, BK virus-based vector leads to an unusual phenotype: cells grow rapidly, forming densely packed, multilayered foci. They progressively form syncytia, some containing as many as six nuclei, and ultimately lyse 1 month after transfection. Some of these properties are reminiscent of oncogenically transformed cells. Cells transfected with the identical vector expressing a variant of eIF-4E, which contains alanine at position 53 and thus cannot be phosphorylated at the major in vivo site, grow normally. Estimations using the Ala-53 variant or a bacterial chloramphenicol acetyltransferase reporter gene in the same vector indicate that the degree of eIF-4E overexpression is 3- to 9-fold more than the endogenous level. These results suggest that eIF-4E may play a key role in cell cycle progression.
MyoD induces growth arrest independent of differentiation in normal and transformed cells.
Authors: Crescenzi M , Fleming T P , Lassar A B , Weintraub H , Aaronson S A
Published: May 2006

MyoD is a gene involved in the control of muscle differentiation. We show that MyoD causes growth arrest when expressed in cell lines derived from tumors or transformed by different oncogenes. MyoD-induced growth inhibition was demonstrated by reduction in the efficiency of colony formation and at the single-cell level. We further show that MyoD growth inhibition can occur in cells that are not induced to activate muscle differentiation markers. The inhibitory activity of MyoD was mapped to the same 68-amino acid segment necessary and sufficient for induction of muscle differentiation, the basic-helix-loop-helix motif. Mutants with alterations in the basic region of MyoD that fail to bind or do not activate a muscle-specific enhancer inhibited growth; mutants with deletions in the helix-loop-helix region failed to inhibit growth. Thus, inhibition of cell growth by MyoD seems to occur by means of a parallel pathway to the one that leads to myogenesis. We conclude that MyoD is a prototypic gene capable of functionally activating intracellular growth inhibitory pathways.
Cell position and developmental fate in leech embryogenesis.
Authors: Keleher G P , Stent G S
Published: May 2006

The o and p blast cell bandlets of the leech Theromyzon rude, which normally produce two different sets of identifiable cells designated the "O" and "P" fates, respectively, form an equivalence group: in embryos experimentally deprived of their p bandlet, the blast cells of the adjacent o bandlet may "transfate" and take on the P fate. Loss of the p bandlet is not, however, a sufficient condition for transfating of the o bandlet. Rather, loss of the p bandlet allows the o bandlet to shift into ectopic positions, and it is the ultimate position of the o bandlet that mandates which fate--O or P--the blast cells will take on. Therefore, the choice of the pluripotent o blast cells to follow either the O or P developmental pathway depends on their perception of positional cues provided by cells outside the equivalence group rather than on a direct interaction with p blast cell equivalence group members.
Dependence of hormone secretion on activation-inactivation kinetics of voltage-sensitive Ca2+ channels in pituitary gonadotrophs.
Authors: Stojilković S S , Iida T , Virmani M A , Izumi S , Rojas E , Catt K J
Published: May 2006

The relationships between the activation status of voltage-sensitive Ca2+ channels and secretory responses were analyzed in perfused rat gonadotrophs during stimulation by high extracellular K+ concentration ([K+]e) or the physiological agonist, gonadotropin-releasing hormone (GnRH). Increase of [K+]e to 50 mM evokes an on-off secretory response, with a rapid rise in luteinizing hormone (LH) secretion to a peak at 35 sec (on response) followed by an exponential decrease to the steady-state level. Cessation of K+ stimulation elicits a transient (off) response followed by an exponential decrease to the basal level. The LH response to high [K+]e is nifedipine-sensitive and its amplitude depends on membrane potential. There is a close relationship between the LH secretory response to high [K+]e and the amplitude of the inward Ca2+ current measured at 100 msec in whole-cell patch clamp experiments. In addition, the profile of the LH secretory response is similar to that of the response of intracellular Ca2+ concentration ([Ca2+]i) in K(+)-stimulated cells. In Ca2(+)-deficient medium, the effect of high [K+]e is abolished; subsequent elevation of [Ca2+]e during the K+ pulse is followed by restoration of the on response, but with reduced magnitude. Agonist stimulation during the steady-state phase of the [K+]e pulse or after repetitive stimulation by high [K+]e elicited biphasic [Ca2+]i and secretory responses with a significantly reduced plateau phase; conversely, K(+)-induced LH release was reduced in cells treated with desensitizing doses of GnRH. These findings indicate that depolarization-induced changes in the status of voltage-sensitive Ca2+ channels determine the profiles of [Ca2+]i and LH responses to stimulation by high [K+]e; the initial activation of dihydropyridine-sensitive Ca2+ channels is clearly dependent on membrane potential, whereas their subsequent inactivation depends on increased [Ca2+]i. Such inactivation of voltage-sensitive Ca2+ channels also occurs during GnRH action and may represent an additional regulatory mechanism to limit the entry of extracellular Ca2+ during prolonged or frequent agonist stimulation.
Domain communication in the dynamical structure of human immunodeficiency virus 1 protease.
Authors: Harte W E , Swaminathan S , Mansuri M M , Martin J C , Rosenberg I E , Beveridge D L
Published: May 2006

A dynamical model for the structure of the human immunodeficiency virus 1 (HIV-1) protease dimer in aqueous solution has been developed on the basis of molecular dynamics simulation. The model provides an accurate account of the crystal geometry and also a prediction of the structural reorganization expected to occur in the protein in aqueous solution compared to the crystalline environment. Analysis of the results by means of dynamical cross-correlation coefficients for atomic displacements indicates that domain-domain communication is present in the protein in the form of a molecular "cantilever" and is likely to be involved in enzyme function at the molecular level. The dynamical structure also suggests information that may ultimately be useful in understanding and further development of specific inhibitors of HIV-1 protease.
Lovastatin decreases coenzyme Q levels in rats.
Authors: Willis R A , Folkers K , Tucker J L , Ye C Q , Xia L J , Tamagawa H
Published: May 2006

Lovastatin is used for the treatment of hypercholesterolemia. It functions by inhibiting the enzyme, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.34), that is required for the conversion of 3-hydroxy-3-methylglutaryl-coenzyme A to mevalonic acid. Since biosynthesis of both cholesterol and coenzyme Q (CoQ) requires mevalonic acid as a precursor, it was considered that lovastatin therapy would also result in a lowering of cellular CoQ levels. This study was conducted to determine whether lovastatin treatment does decrease CoQ levels and whether such decreases can be prevented by CoQ supplementation. Forty-five adult male Holtzman rats were randomly assigned to one of three treatment groups. Controls were fed ground laboratory rat chow ad libitum. The other two groups were fed ground laboratory rat chow containing 400 mg of lovastatin per kg of diet ad libitum. One of the lovastatin-fed groups received CoQ10 (15 mg per kg of body weight) daily via stomach intubation. After 4 weeks, samples of heart, liver, and blood were analyzed for CoQ concentrations. Results indicated that CoQ concentrations in all tissues analyzed were decreased in lovastatin-treated rats. Lovastatin-treated animals that were supplemented with CoQ10 had blood, heart, and liver CoQ10 concentrations that approximated or exceeded those of control animals. It is concluded that lovastatin does indeed lower tissue concentrations of CoQ and that a return to normal can be achieved by supplementation with CoQ.
Posttranslationally processed structure of the human platelet protein smg p21B: evidence for geranylgeranylation and carboxyl methylation of the C-terminal cysteine.
Authors: Kawata M , Farnsworth C C , Yoshida Y , Gelb M H , Glomset J A , Takai Y
Published: May 2006

smg p21A and -B are small GTP-binding proteins that share putative effector and consensus C-terminal sequences with ras p21 proteins. In the present report, we showed that human platelet smg p21B became labeled when intact platelets were incubated with exogenous [3H]mevalonolactone and when a purified preparation of smg p21B was incubated with bovine brain membranes and S-adenosyl-L-[methyl-3H]methionine. In addition, we demonstrated by gas chromatography/mass spectrometry that treatment of smg p21B with Raney nickel released a geranylgeranyl moiety in a molar ratio of about 1:1. In contrast, treatment of smg p21B with NH2OH or KOH yielded no evidence for the presence of a palmitoyl thioester. Extensive digestion of smg p21B with Achromobacter protease I yielded two C-terminal tripeptides that contained serine and cysteine in a molar ratio of 2:1. Both peptides were modified by a thioether-linked geranylgeranyl group. One of the peptides comigrated with a 3H-labeled proteolytic product of methylated smg p21B on reverse-phase HPLC and this peptide appeared at the same retention time as that of the other peptide after being treated with KOH. Since the cDNA-predicted C-terminal sequence of smg p21B contains a unique Ser-Ser-Cys peptide within its C-terminal domain, -Lys-Lys-Ser-Ser-Cys-Gln-Leu-Leu184, these results indicate that smg p21B is posttranslationally modified by geranylgeranylation of Cys-181 and suggest that further modifications cause proteolytic removal of the three predicted C-terminal amino acids followed by partial methylation of the cysteinyl carboxyl group.
Rabbit ileal villus cell brush border Na+/H+ exchange is regulated by Ca2+/calmodulin-dependent protein kinase II, a brush border membrane protein.
Authors: Cohen M E , Reinlib L , Watson A J , Gorelick F , Rys-Sikora K , Tse M , Rood R P , Czernik A J , Sharp G W , Donowitz M
Published: May 2006

Ileal brush border membranes contain an endogenous Ca2+/calmodulin (CaM)-dependent protein kinase activity that modulates the activity of the apical membrane Na+/H+ exchanger. To further characterize this kinase, synapsin I, a substrate for Ca2+/CaM-dependent protein kinases, was added to preparations of ileal brush border membranes. In the presence of Ca2+/CaM, synapsin I was phosphorylated. Phosphopeptide mapping demonstrated that the addition of Ca2+/CaM to brush border membranes stimulated the phosphorylation of sites in synapsin I specific for Ca2+/CaM-dependent protein kinase II. Immunoblots containing brush border and microvillus membrane proteins were probed with an antibody that recognizes the 50-kDa subunit of rat brain Ca2+/CaM-dependent protein kinase II. This antibody labeled major and minor species of 50 and 53 kDa, respectively, with more labeling of the brush border than the microvillus membranes. Right-side-out ileal villus cell brush border vesicles were prepared containing CaM, ATP, and 350 nM free Ca2+. Na+/H+ exchange was inhibited by the presence of Ca2+/CaM/ATP within the vesicles. A 21-amino acid peptide inhibitor of CaM kinase II was enclosed within some vesicle preparations by freeze-thaw. The effect on Na+/H+ exchange of Ca2+/CaM/ATP was partially reversed by the inhibitor peptide. These studies demonstrate the presence of Ca2+/CaM-dependent protein kinase II in rabbit ileal villus cell brush border membranes. Based on the effect of a specific inhibitor peptide of Ca2+/CaM kinase II, it is concluded that this kinase inhibits brush border Na+/H+ exchange, which participates in the regulation of ileal Na+ absorption.
Molecular cloning of an olfactory gene from Drosophila melanogaster.
Authors: Hasan G
Published: May 2006

An olfactory gene olfE, which affects response to benzaldehyde in larvae and adults of Drosophila melanogaster, has been mapped between two breakpoints on the X chromosome. The breakpoints have been shown to lie at a distance no greater than 25 kilobases (kb). A 14-kb genomic fragment from this region has been used for germ-line transformation of olfE mutant flies, and in one of three transformant lines obtained, rescue of the olfE phenotype is observed by two separate behavioral assays. Transcript analysis of the region that rescues the olfE phenotype has shown one major transcript at 5.4 kb and a minor one at 1.7 kb. Both of these transcripts are probably alternatively spliced products of the olfE gene. A developmental and tissue-specific profile of the 5.4-kb olfE message has shown that it is present at all developmental stages, suggesting that the gene may be multifunctional.
Increased conversion of phosphatidylinositol to phosphatidylinositol phosphate in Dictyostelium cells expressing a mutated ras gene.
Authors: Van der Kaay J , Draijer R , Van Haastert P J
Published: May 2006

Dictyostelium discoideum cells that overexpress a ras gene with a Gly12----Thr12 mutation (Dd-ras-Thr12) have an altered phenotype. These cells were labeled with [3H]inositol and the incorporation of radioactivity into inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was analyzed and found to be higher than in control cells. In contrast, the total mass of Ins(1,4,5)P3, as assessed with an assay using a specific Ins(1,4,5)P3-binding protein, was not significantly different between control and Dd-ras-Thr12 cells. Cells were labeled with [3H]inositol and the incorporation of radioactivity in all inositol metabolites was analyzed. Increased levels of radioactivity were observed for phosphatidylinositol phosphate (PtdInsP), phosphatidylinositol bisphosphate (PtdInsP2), Ins(1,4,5)P3, inositol 1,4-bisphosphate, inositol 4,5-bisphosphate, and inositol 4-monophosphate in Dd-ras-Thr12 cells relative to control cells. Decreased levels were found for phosphatidylinositol (PtdIns) and inositol 1-monophosphate. Calculations on the substrate/product relationships [i.e., Ins(1,4,5)P3/PtdInsP2] demonstrate that the observed differences are due only to the increased conversion of PtdIns to PtdInsP; other enzyme reactions, including phospholipase C, are not significantly different between the cell lines. The activity of PtdIns kinase in vitro is not different between Dd-ras-Thr12 and control cells, suggesting that either the regulation of this enzyme is altered or that the translocation of substrate from the endoplasmic reticulum to the kinase in the plasma membrane is modified. The results suggest multiple metabolic compartments of Ins(1,4,5)P3 in Dictyostelium cells. In Dd-ras-Thr12 transformants the increased conversion of PtdIns to PtdInsP leads to increased levels of Ins(1,4,5)P3 in the compartment with a high metabolic turnover. This Ins(1,4,5)P3 compartment is suggested to be involved in the regulation of cytosolic Ca2+ levels.
Mitochondrial respiratory inhibition by N-methylated beta-carboline derivatives structurally resembling N-methyl-4-phenylpyridine.
Authors: Albores R , Neafsey E J , Drucker G , Fields J Z , Collins M A
Published: May 2006

Mitochondrial accumulation and respiratory inhibition are critical steps in the actions of N-methyl-4-phenylpyridinium ion (MPP+), the toxic metabolite of the parkinsonism-inducing agent, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. We examined the respiratory characteristics of 2-methylated beta-carbolines (2-Me beta Cs) and 2-methylated 3,4-dihydro-beta-carbolines (2-MeDH beta Cs), which encompass the MPP+ structure. As indoleamine derivatives, they could have endogenous roles in idiopathic parkinsonism. With rat liver mitochondria, the order for inhibition of NAD(+)-linked O2 consumption (6-min preincubations) was as follows: MPP+ = 2-methylharmine greater than 2-methylharmol = 2-methylharmaline much greater than 2-methylharmalol greater than 2-methylnorharman greater than 6-OH-2-methylharmalan much greater than 2-methylharman. Similar to MPP+, 2-MeDH beta C/2-Me beta C inhibition was potentiated by tetraphenylboron and reversed by dinitrophenol, consistent with the involvement of cationic forms. However, the participation of neutral forms was indicated by the 2-MeDH beta C/2-Me beta C inhibitory time courses, which were unlike MPP+. The neutral forms probably arise via indolic nitrogen deprotonation because the characteristics of a cationic beta-carboline that cannot N-deprotonate, 2,9-dimethylnorharman, mirrored MPP+ rather than 2-Me beta Cs. Succinate-supported respiration was also significantly blocked by 2-MeDH beta Cs/2-Me beta Cs, but results with tetraphenylboron and 2,9-dimethylnorharman indicated that cationic forms were less important than in the inhibition of NAD(+)-linked respiration. We suggest that the relatively potent inhibition by certain 2-MeDH beta Cs/2-Me beta Cs involves neutral forms for passive mitochondrial entry and cationic as well as neutral forms that act at several respiratory sites. Respiratory inhibition could reasonably underlie the reported neurotoxicity of 2-Me beta Cs.
A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism.
Authors: Morrow J D , Hill K E , Burk R F , Nammour T M , Badr K F , Roberts L J
Published: May 2006

Increasing attention has focused on the role of free radicals derived from oxygen in the pathophysiology of a wide variety of disorders. One of the well-recognized targets of free radical-induced injury is peroxidation of lipids. Using a variety of approaches, we have found that a series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase mechanism involving free radical-catalyzed peroxidation of arachidonic acid. Levels of these compounds in normal human plasma and urine range from 5 to 40 pg/ml and 500 to 4000 pg/mg of creatinine, respectively. In rats, their formation was found to increase as much as 200-fold in association with marked free radical-catalyzed lipid peroxidation induced by administration of CCl4 and diquat. To explore whether these prostanoids can exert biological activity, the effects of one of the compounds formed by this mechanism, 8-epi-prostaglandin F2 alpha, was examined in the kidney in the rat. Infusion of 8-epi-prostaglandin F2 alpha into a peripheral vein (5 micrograms/kg per min) or intrarenally (0.5-2.0 micrograms/kg per min) resulted in marked parallel reductions in renal blood flow and glomerular filtration rate. That the formation of these prostanoids is catalyzed by free radicals and that they can exert potent biological activity suggest that these prostanoids may participate as pathophysiological mediators in oxidant injury. Quantification of these compounds may also provide a noninvasive approach to assess oxidant status in humans. That the formation of these prostanoids occurs independent of the catalytic activity of the cyclooxygenase enzyme suggests that there may be limitations at times regarding the reliability of the use of cyclooxygenase inhibitors to assess the role of prostaglandins in certain pathophysiological processes.