bluuebunny/crossref_metadata_2025 huggingface dataset:
Internal duplication and sequence homology in factors V and VIII.
Authors: Fass D N , Hewick R M , Knutson G J , Nesheim M E , Mann K G
Published: May 2006

Blood coagulation factors V and VIII each serve cofactor functions with different vitamin K-dependent serine proteases of the coagulation cascade. Physical, physiologic, and kinetic data suggest analogous structures and functions for these two proteins. Proteolytically activated factor V (factor Va) is required for the efficient production of thrombin from prothrombin by factor Xa. Similarly, activated factor VIII (factor VIIIa) performs its cofactor activity with factor IXa to produce the activated form of factor X (factor Xa). The studies reported here on the sequences from the thrombin-activated and unactivated cofactors provide evidence that factor V and factor VIII are chemically related and that the structures of both cofactors involve some tandem duplication.
Glucose transport protein is structurally and immunologically related to band 3 and senescent cell antigen.
Authors: Kay M M
Published: May 2006

Senescent cell antigen, a polypeptide that appears on the surface of senescent and damaged cells, has been shown to be derived from band 3. In the present study, the relationship between the as yet unidentified glucose transporter and senescent cell antigen is examined. Since cytochalasin B is a specific and potent competitive inhibitor of glucose transport in human erythrocytes, the glucose transport carrier was isolated by affinity chromatography on cytochalasin B-Sepharose 4B columns and eluted with D-glucose. This purification procedure is both a method of isolation and a functional assay for the glucose transporter. Purified glucose transporter gave a sharp, single band at Mr approximately equal to 60,000 when analyzed by NaDod-SO4/PAGE. Glucose transporter was identified in erythrocyte membranes by the immunoblotting technique, using both antibodies raised against purified glucose transporter and anti-idiotypic antibodies. Two-dimensional peptide mapping revealed substantial peptide homology between glucose transporter and senescent cell antigen. In addition, the glucose transporter shared peptide homology with band 3 and its defined proteolytic fragments located toward the carboxyl terminus of band 3. Peptide homology between glucose transporter and the Mr approximately equal to 41,000 cytoplasmic segment of band 3 that contains the amino terminus could not be demonstrated. Thus, glucose transporter appears to be part of or derived from band 3, and to share substantial peptide homology with senescent cell antigen.
Allelic polymorphism and transassociation of molecules encoded by the HLA-DQ subregion.
Authors: Giles R C , DeMars R , Chang C C , Capra J D
Published: May 2006

A monoclonal antibody, CC11.23, with monomorphic specificity predominantly for products of the HLA-DQ subregion, has been used to demonstrate primary structural variation among DQ molecules. Two cell lines of each haplotype (DR1-7) were radiolabeled with [3H]tyrosine. alpha and beta chains were isolated from CC11.23-reactive preparations, and their amino-terminal tyrosine sequences were determined. Each DR haplotype (with the exception of DRw6) was found to express a distinct DQ molecule with a minimum of three allelic forms of the DQ alpha chain and five allelic forms of the DQ beta chain. At the primary structural level, the locus for the DQ beta chain appears to be as polymorphic as the locus for the DR beta chain. Unlike the locus for the DR alpha chain (which is essentially nonpolymorphic), the locus for the DQ alpha chain was found to be polymorphic. Comparison of DQ molecules from two different heterozygous cell lines with those from homozygous cell lines revealed that in heterozygotes, DQ alpha chains from either allele can associate with DQ beta chains from one allele. The formation of hybrid HLA-DQ molecules by both cis and trans gene complementation, coupled with several polymorphic forms of each of the DQ subunits, considerably increases the repertoire of DQ alloantigens in heterozygotes.
A human acetylcholinesterase gene identified by homology to the Ace region of Drosophila.
Authors: Soreq H , Zevin-Sonkin D , Avni A , Hall L M , Spierer P
Published: May 2006

The Ace locus of the Drosophila genome controls biosynthesis of the neurotransmitter-hydrolyzing enzyme acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). We injected the mRNA species hybridizing with DNA fragments from this region into Xenopus oocytes, in which acetylcholinesterase mRNA is translated into active acetylcholinesterase. A 2.0-kilobase (kb) fragment of DNA from this region selectively hybridizes with Drosophila mRNA capable of inducing the biosynthesis of acetylcholinesterase in oocytes. This Drosophila DNA fragment cross-hybridized with human brain poly(A)+ RNA. We therefore used this DNA fragment as a probe for homologous sequence(s) in a human genomic DNA library and thus selected a 13.5-kb human DNA segment. DNA blot-hybridization revealed that a 2.6-kb fragment of this human DNA segment hybridizes with the Drosophila 2.0-kb DNA fragment. Both Drosophila and human fragments hybridized with a human brain mRNA species of about 7.0-kb that was barely detectable in the acetylcholinesterase-deficient HEp carcinoma. A fraction containing mRNA of similar size, extracted from human brain, induced acetylcholinesterase biosynthesis in oocytes. The human DNA fragment also was used in hybridization-selection experiments. In oocytes, hybrid-selected human brain mRNA induced acetylcholinesterase activity that was completely inhibited by 1,5-bis[4-allyldimethylammonium)phenyl]pentan-3-one dibromide but not by tetraisopropyl pyrophosphamide, a differential response to these inhibitors characteristic of "true" human brain acetylcholinesterase. These findings strongly suggest that both the Drosophila and the human DNA fragments are directly involved in controlling acetylcholinesterase biosynthesis.
Prenatal expression of a lethal genetic defect in carbohydrate metabolism in mice.
Authors: Tyson F L , Essien F B
Published: May 2006

Mouse fetuses homozygous for the lethal cab (cardiac abnormal) mutation are characterized by pleiotropic effects that lead to immediate postnatal death. Mutant fetuses have only 4% of the normal amount of hepatic glycogen and 39% of the normal cardiac glycogen reserve, coupled with lower specific activities of glycogen synthase and phosphorylase. Analysis with the periodic acid-Schiff reagent histochemical stain demonstrated that cab homozygotes also have reduced amounts of structural polysaccharides. One of the most distinctive mutant phenotypic traits is severe prenatal hypoglycemia, with average (+/-SEM) plasma glucose concentrations of 0.35 +/- 0.14 mM in late fetuses compared to 3.47 +/- 0.69 mM in normal littermates. Compromise of glucose transport from dam to fetus or altered cellular glucose utilization was considered as a possible basis for the low extracellular and intracellular (hepatic) levels of glucose in mutants. Transport of the glucose analogue alpha-methyl[14C]glucoside by the placenta of cab homozygotes is normal. However, metabolism of [14C]glucose by mutant cells yields only 20% of the normal amount of 14CO2. This reduced efficiency of glucose metabolism is correlated with lower ATP concentrations in mutant organs. Aberrant glucose utilization may account for the pleiotropic features of the cab syndrome.
Laminin is associated with the "neurite outgrowth-promoting factors" found in conditioned media.
Authors: Lander A D , Fujii D K , Reichardt L F
Published: May 2006

Conditioned media (CMs) from many cell types contain a factor that can adsorb to a polycationic substratum and cause neurons plated on that substratum to extend neurites rapidly and profusely. The extracellular matrix glycoprotein laminin, when bound to a substratum, elicits a similar response by neurons. In this report, six CMs that contain a "neurite outgrowth-promoting factor" were studied. Immunoprecipitation with affinity-purified anti-laminin antibodies demonstrated that laminin is present in all of them, and when it was selectively removed, there was a corresponding loss of neurite outgrowth-promoting activity in each CM. Antibodies to purified laminin failed, however, to block the outgrowth-promoting activity of five of the CMs tested, even though these antibodies blocked the outgrowth-promoting activity of purified laminin in the same assay. This result could reflect differences in amino acid sequence or protein modification between CM-derived laminin and the purified laminin used in generating antibodies. Alternatively, it could reflect the fact that other molecules bind to laminin in CMs and could interfere with the binding of antibodies to sites on laminin that are important for biological activity.
Multiply drug-resistant human KB carcinoma cells have decreased amounts of a 75-kDa and a 72-kDa glycoprotein.
Authors: Richert N , Akiyama S , Shen D , Gottesman M M , Pastan I
Published: May 2006

Human KB carcinoma cells were selected in sequential steps for resistance to colchicine and found to be cross-resistant to multiple drugs, including vinblastine, adriamycin, and actinomycin D. Compared with the parental line, the multiply resistant cells have decreased amounts of two [35S]methionine-labeled proteins with apparent molecular masses of 75 and 72 kDa. These proteins reappear in a revertant, drug-sensitive cell line. Both proteins are labeled with [14C]glucosamine and are retained on a wheat germ agglutinin-agarose column, indicating that they are glycoproteins. These data suggest that in this human cell line, these two glycoproteins can serve as a marker of the multiple drug-resistance phenotype and may play a role in its etiology.
Pattern analysis of 5S rRNA.
Authors: Eigen M , Lindemann B , Winkler-Oswatitsch R , Clarke C H
Published: May 2006

Some 200 different 5S rRNA sequences from eubacteria, chloroplasts, mitochondria, archaebacteria, and eukaryotes were analyzed for evolutionary kinship relationships and associated sequential features. Group-specific occupation schemes for the 149 positions of an overall alignment were established. Eubacterial, archaebacterial, and intermediate occupation schemes all yield a strongly biased base triplet pattern in one of the three possible reading frames strongest for eubacterial, chloroplastic, and archaebacterial, but still detectable for mitochondrial and eukaryotic cytoplasmic sequences. The frequency of triplets decays in the order RNY greater than RNR greater than YNY greater than YNR; R being a purine (guanine or adenine), Y is a pyrimidine (cytosine or uracil), and N is any base. A strong preference for guanine or cytosine was found in all triplet positions. The effects show no exceptions and are clearly above the level of statistical fluctuations.
A lethal deletion on mouse chromosome 7 affects regulation of liver-cell-specific functions: posttranscriptional control of serum protein and transcriptional control of aldolase B synthesis.
Authors: Sala-Trepat J M , Poiret M , Sellem C H , Bessada R , Erdos T , Gluecksohn-Waelsch S
Published: May 2006

Steady-state levels of mRNAs were determined for the serum proteins albumin, alpha-fetoprotein (AFP), and transferrin, as well as for aldolase B in livers of newborn mice homozygous for a radiation-induced lethal deletion (c14CoS) in chromosome 7. Deficiencies in synthesis and secretion of the serum proteins as well as in activities of certain liver-specific enzymes characterize these homozygotes. The results of RNA dot and gel-blot hybridizations with the respective cloned cDNA probes showed a decrease to one-fourth of aldolase B mRNA levels in homozygous mutant livers compared to normal littermates, in contrast to normal levels of mRNA sequences for the three serum proteins in the mutants. Furthermore, the mRNA sequences were shown to be present as mature mRNA molecules in both mutant and normal littermate livers. We suggest that the deficiencies of liver-specific serum proteins and those of the enzymes caused by the lethal deletions around the albino locus on chromosome 7 of the mouse are due to different causes. In the case of the liver-specific enzyme examined here--i.e., aldolase B--control at the level of transcription or of message stability is affected in the homozygous deletion mutants, whereas the deficiencies of serum proteins are not reflected on the mRNA level and owe their origin to an effect on a posttranscriptional or translational level. These results lend further support to the assumption that the deleted portion of the genome includes genes concerned with the control and regulation of liver cell differentiation.
B-cell growth factor (B-cell growth factor I or B-cell-stimulating factor, provisional 1) is a differentiation factor for resting B cells and may not induce cell growth.
Authors: Oliver K , Noelle R J , Uhr J W , Krammer P H , Vitetta E S
Published: May 2006

B-cell growth factor I [BCGF I or B-cell-stimulating factor, provisional 1 (BSFp1)] has been defined as a T-cell-derived lymphokine that acts as a co-stimulator of polyclonal B-cell growth in B cells cultured with anti-mu, anti-delta, or anti-Ig. Based on a number of studies it has been suggested that anti-Ig induces cell enlargement, entry into the G1 phase of the cell cycle, and expression of receptors for BSFp1. BSFp1 then induces entry of the cells into S phase. By adding BSFp1 prior to anti-Ig, we have found evidence that BSFp1 renders cells susceptible to anti-Ig-mediated entry of cells into G2/S phase. In contrast, if cells are first treated with anti-Ig, washed, and then cultured with BSFp1, they do not enter S phase. Taken together, these results suggest that BSFp1 acts on the resting B cells not as a growth factor but rather as a lymphokine that prepares cells for anti-Ig-mediated activation. Taken together with previous reports that BSFp1 induces increased expression of Ia antigens on resting B cells, these studies suggest that BSFp1 may be a differentiation factor rather than a growth factor and that it acts on resting B cells.
Plasma and cellular retinoid-binding proteins and transthyretin (prealbumin) are all localized in the islets of Langerhans in the rat.
Authors: Kato M , Kato K , Blaner W S , Chertow B S , Goodman D S
Published: May 2006

The immunohistochemical localization of plasma retinol-binding protein (RBP), cellular retinol-binding protein (CRBP), and transthyretin (TTR) was studied in rat pancreas. The studies employed antibodies purified by immunosorbent affinity chromatography, permitting the specific staining and localization of each antigen by the unlabeled peroxidase-antiperoxidase method. Specific immunostaining for each of these three proteins was found localized to the islets of Langerhans. Both RBP and CRBP were localized in cells that were peripherally distributed within the islets, with an anatomic distribution that resembled that of the glucagon-containing A cells. Immunoreactive TTR was localized in cells that were more centrally distributed in the islets, with an anatomic distribution that resembled that of the insulin-containing B cells. These findings were confirmed by radioimmunoassay of a homogenate of isolated rat islets. By using sensitive and specific radioimmunoassays for each antigen, unusually high levels of CRBP, RBP, TTR, and cellular retinoic acid-binding protein (CRABP) were found in rat islets. The physiological significance of the localization of RBP, CRBP, CRABP, and TTR in the islets is not known. The findings suggest that retinoids and their binding proteins may play important metabolic roles within islet cells, and hence that they may be involved in some way in the biological, endocrine, function of the islets.
Selective enhancement of the expression of granulocyte functional antigens 1 and 2 on human neutrophils.
Authors: Vadas M A , López A F , Williamson D J
Published: May 2006

The regulation of expression of two human granulocyte functional antigens (GFA-1 and GFA-2) was examined. N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) caused a rapid, dose-dependent enhancement of the expression of these antigens, 2- to 4-fold within 30 min, but not of another surface structure, beta 2-microglobulin. Pretreatment of the cells with cytochalasin B at 5 micrograms/ml further enhanced the effect of fMet-Leu-Phe on the expression of GFA-2, raising its surface expression 11-fold. Lipopolysaccharide also stimulated the expression of GFA-1 and GFA-2. The effect of lipopolysaccharide was less than that of fMet-Leu-Phe and was more marked on GFA-1 than on GFA-2. Pretreatment of neutrophils with fMet-Leu-Phe not only stimulated their cytotoxic activity against antibody-coated target cells but also increased their capacity to be stimulated by monoclonal antibodies to GFA-1 and GFA-2. These findings show that the expression of functional surface structures on human neutrophils is subject to rapid and selective regulation.
Differential regulation of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase isoenzymes by cyclic AMP-dependent protein kinase and calmodulin-dependent phosphatase.
Authors: Sharma R K , Wang J H
Published: May 2006

Purified bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) contains isozymes that are composed of two distinct subunits with molecular masses of 60,000 and 63,000 daltons. Analysis by NaDodSO4 gel electrophoresis and autoradiography of a phosphodiesterase sample phosphorylated in the presence of [32P]ATP and bovine heart cAMP-dependent protein kinase catalytic subunit revealed that only the 60-kDa subunit was phosphorylated. By using an isozyme preparation greatly enriched with the 60-kDa subunit, the following observations regarding the subunit phosphorylation were made. First, the phosphorylation resulted in the maximal incorporation of about 2 mol of phosphate per mol of subunit. Second, complete inhibition of 60-kDa subunit phosphorylation was approached at a saturating concentration of Ca2+ when a molar ratio of calmodulin to phosphodiesterase of 2:1 was used. No inhibition was observed in the presence of either Ca2+ or calmodulin alone. Third, the phosphorylation was accompanied by a decrease in the enzyme affinity for calmodulin; calmodulin concentrations required for 50% activation of nonphosphorylated and maximally phosphorylated phosphodiesterase isozyme samples were 0.51 and 9.3 nM, respectively. Fourth, the phosphodiesterase isozyme could be dephosphorylated by the calmodulin-dependent phosphatase (calcineurin) in the presence of Ni2+ or Mn2+, the dephosphorylation being associated with an increase in the enzyme affinity for calmodulin. Fifth, peak II rabbit liver phosphoprotein phosphatase catalytic unit did not catalyze the dephosphorylation of the phosphodiesterase isozyme.
Antifreeze protein genes are tandemly linked and clustered in the genome of the winter flounder.
Authors: Scott G K , Hew C L , Davies P L
Published: May 2006

We have used genomic Southern blots and restriction maps of genomic clones to examine the organization of the antifreeze protein multigene family in the winter flounder. The majority of the approximately equal to 40 antifreeze protein (AFP) genes in this fish are present in 7- to 8-kilobase-pair (kbp) elements of DNA, which are iterated as tandem direct repeats. Each repeat contains a single antifreeze protein gene that is 1 kbp long, and all of these genes have the same transcriptional orientation. Although the repeated elements are highly homologous, they do show some restriction site and restriction length polymorphisms. When flounder genomic DNA is digested with restriction endonucleases that do not cut within the repeats, most of the antifreeze protein genes reside in fragments that are at least 40 kbp long, representing clusters of five or more repeats in tandem. After genomic DNA is digested with Xba I or Xho I, these genes are present in fragments of exceptionally high molecular weight, suggesting that the clusters themselves are grouped together in the genome. The AFP gene locus may have evolved by gene amplification as recently as 10(6) years ago in response to the onset of the Cenozoic ice age.
Molecular cloning of a full-length cDNA for human alcohol dehydrogenase.
Authors: Ikuta T , Fujiyoshi T , Kurachi K , Yoshida A
Published: May 2006

We have cloned a full-length cDNA coding for human alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) from a human liver cDNA library constructed in phage lambda gt11. The library was screened by using a rabbit antibody against human ADH as a first probe, by the modified method of Young and Davis [Young, R. A. & Davis, R. W. (1983) Proc. Natl. Acad. Sci. USA 80, 1194-1198]. Mixed 14-mer synthetic oligonucleotides encoding Asp-Asp-His-Val-Val and Gln-Cys-Gly-Lys-Cys were used as a second probe. These amino acid sequences are considered to be common in all three subunits (alpha, beta, and gamma) controlled by the ADH1, ADH2, and ADH3 loci. Ten lambda gt11 recombinants of 35 positive plaques obtained by antibody screening contained inserted cDNAs of 1.5-2.4 kilobase pairs and were found to exhibit positive signals by hybridization with synthetic probes. One of them, with an inserted cDNA of 1631 base pairs, contained a sequence that encodes 374 amino acid residues of the human beta 1 subunit, a chain initiation codon, a chain termination codon, and additional 3' and 5' untranslated regions. A complete amino acid sequence of the human beta 1 subunit was deduced from the cDNA.
Nucleotide sequence of two overlapping myc-related genes in avian carcinoma virus OK10 and their relation to the myc genes of other viruses and the cell.
Authors: Hayflick J , Seeburg P H , Ohlsson R , Pfeifer-Ohlsson S , Watson D , Papas T , Duesberg P H
Published: May 2006

Avian carcinoma virus OK10 has the genetic structure gag-delta pol-myc-delta env. It shares the transformation-specific myc sequence with three other avian carcinoma viruses (MC29, MH2, CMII) and also with a normal chicken gene proto-myc and the gag, pol, and env elements with non-transforming retroviruses. Unlike the other myc-containing viruses, which synthesize singular myc proteins, OK10 synthesizes two different myc-related proteins of 200 and 57 kDa. Here we have sequenced the myc region of an infectious OK10 provirus to investigate how OK10 synthesizes two different proteins from the same myc domain and to identify characteristic differences between the normal proto-myc gene and the myc-related viral transforming genes. It was found that the 1.6-kilobase myc domain of OK10 is colinear and coterminal with the myc domains of MC29, MH2, and the terminal two exons of proto-myc. It is preceded by the same splice acceptor as the myc sequence of MH2 and as the second proto-myc exon. From this and the known structure of retroviruses, it follows that the OK10 gene encoding the 57-kDa protein is discontinuous with a small 5' exon that includes six gag codons and a large 3' myc exon (delta gag-myc). This gene and the delta gag-myc gene of MH2 are isogenic. The proto-myc-derived intron preceding the myc domain of OK10 is in the same reading frame as the adjacent delta pol and myc domains and, hence, is part of the gag-delta pol-myc gene encoding the 200-kDa protein. Sequence comparisons with proto-myc and MC29 and MH2 indicate that there are no characteristic mutations that set apart the viral myc domains from proto-myc. It is concluded that transforming function of viral myc-related genes correlates with the lack of a viral equivalent of the first proto-myc exon(s) and conjugation of the viral myc domains with large or small retroviral genetic elements rather than with specific point mutations. Because OK10 and MH2 each contain two genes with potential transforming function (namely, delta gag-myc and gag-delta pol-myc or delta gag-mht, respectively), it remains to be determined whether the delta gag-myc genes have transforming function on their own or need helper genes. The possible helper requirement cannot be very specific because the two potential helper genes are very different.
19-Hydroxylation of androgens in the rat brain.
Authors: Hahn E F , Miyairi S , Fishman J
Published: May 2006

Aromatization of androgens in the central nervous system is linked with sexual differentiation of the brain and, thus, determines the nature of sexual behavior and the control of gonadotropin secretion. The process of aromatization, as determined in the human placenta, proceeds through two successive hydroxylations at C-19, the products of which are then virtually completely converted via a third hydroxylation at C-2 to estrogens. We now report that in the rat brain, 19-hydroxylation of androgens greatly exceeds aromatization and the 19-hydroxy- and 19-oxoandrogen products accumulate in quantities 5 times greater than the estrogens. This relationship implies that the aromatization sequence in the brain is deficient in the terminal hydroxylase, and the process is distinct from that in other tissues. The function of 19-hydroxy- and 19-oxotestosterone in the central nervous system is unknown but, unlike the reduced or aromatized metabolites of the male hormone, these substances cannot be delivered from the circulation and their presence in the brain is totally dependent on in situ formation, making them logical candidates for modulators of neuronal functions.
Specificity analysis of mouse monoclonal antibodies defining cell surface antigens of human renal cancer.
Authors: Finstad C L , Cordon-Cardo C , Bander N H , Whitmore W F , Melamed M R , Old L J
Published: May 2006

Six mouse monoclonal antibodies (mAbs) defining separate systems of cell surface antigens of cultured human renal cancer were tested for reactivity with normal fetal and adult tissues and with neoplastic tissues. Five of the mAbs identified glycoproteins of Mr 160,000 (designated S4), Mr Mr 140,000 (F23), Mr 120,000 (S23 and S27), and Mr 115,000 (S22). The glycoprotein component of Mr 120,000 has been shown recently to be the adenosine deaminase binding protein (ADA-BP) and mAbS23 and mAbS27 define two distinct epitopes on ADA-BP. S22 was not detected on any normal fetal or adult tissues but was found on a subset of renal cancers. S4, F23, S23, and S27 defined distinct domains of the nephron: glomerulus (S4), proximal tubules (S4, F23, S23, and S27), and portions of Henle's loop (S23 and S27). mAbS4 also reacted with the interstitial matrix in the renal medulla and of other tissues, and mAbF23 reacted with fetal and adult fibroblasts. The S23 epitope of ADA-BP was expressed by placental trophoblasts and epithelial cells of breast, prostate, lung, and colon, whereas the S27 epitope was detected on a more limited range of cell types (trophoblasts and prostate epithelium). A panel of 20 renal cell carcinomas was typed for expression of these antigens; 7 phenotypes could be distinguished, with the S4+/F23+/S23+/S27+/S22+ or - phenotype (15 cases) being most common. The other antigenic system, V1, identified a heat-stable antigen that was widely expressed on cultured cell types but showed a restricted pattern of reactivity in tissues. V1 expression was limited to the adrenal cortex, Leydig cells, and the theca of ovarian follicles, and to adrenal cortical carcinomas.
In vitro studies of 2,4-dihydroxyphenylalanine, a prodrug targeted against malignant melanoma cells.
Authors: Morrison M E , Yagi M J , Cohen G
Published: May 2006

We have evaluated the chemotherapeutic potential of 2,4-dihydroxyphenylalanine, a targeted prodrug that can be hydroxylated by tyrosinase (monophenol monooxygenase, EC 1.14.18.1) within melanoma cells to form the cellular toxin 2,4,5-trihydroxyphenylalanine (6-hydroxydopa). 2,4-Dihydroxyphenylalanine proved to be cytotoxic to both B-16 and Cloudman melanoma cells in vitro. The immediate effects of 2,4-dihydroxyphenylalanine included inhibition of DNA, RNA, and protein syntheses. In contrast, no decrease in macromolecular synthesis or viability was seen against cultures of MJY-alpha mammary tumor or L-1210 leukemia, two cell types that do not contain tyrosinase. Within the melanoma cultures, greater cytotoxicity was seen against melanotic (tyrosinase-containing) cells than against amelanotic (tyrosinase-lacking) cells. The cytotoxicity of 2,4-dihydroxyphenylalanine was blocked by 1-phenylthiourea, an inhibitor of tyrosinase. These results show that 2,4-dihydroxyphenylalanine is toxic to melanoma cells and that activation of 2,4-dihydroxyphenylalanine requires the presence of tyrosinase.
An initiator of carcinogenesis selectively and stably inhibits stem cell differentiation: a concept that initiation of carcinogenesis involves multiple phases.
Authors: Scott R E , Maercklein P B
Published: May 2006

A concept of carcinogenesis was recently devised in our laboratory that suggests the development of defects in the control of cell differentiation is associated with an early phase of carcinogenesis. To test this proposal directly, the effects of an initiator of carcinogenesis (i.e., UV irradiation) on proadipocyte stem cell differentiation and proliferation was assayed. In this regard, 3T3 T proadipocytes represent a nontransformed mesenchymal stem cell line that possesses the ability to regulate its differentiation at a distinct state in the G1 phase of the cell cycle as well as the ability to regulate its proliferation at two additional G1 states that are induced by culture in growth factor-deficient or nutrient-deficient microenvironments. The results establish that a low dosage of 254 nm UV irradiation selectively and stably inhibits the differentiation of a high percentage of proadipocyte stem cells without significantly altering their ability to regulate cellular proliferation in growth factor-deficient or nutrient-deficient culture conditions. Differentiation-defective proadipocyte stem cells are demonstrated not to be completely transformed but to show an increased spontaneous transformation rate, as evidenced by the formation of type III foci in high density cell cultures. These data support the role of defects in the control of differentiation in the initiation of carcinogenesis. Other data, however, also suggest that additional cellular defects must be expressed for a cell to be completely initiated. These observations support a concept that the initiation of carcinogenesis involves multiple phases.
Dopamine decreases conductance of the electrical junctions between cultured retinal horizontal cells.
Authors: Lasater E M , Dowling J E
Published: May 2006

Horizontal cells from the white perch were isolated by enzymatic treatment and trituration of the retina and were maintained in culture for 1-5 days. Overlapping pairs of horizontal cells were identified, and the two cells were recorded from simultaneously, using whole-cell patch clamp techniques. Electrical coupling between cells was determined by passing current pulses into one cell, the driver cell, while (i) recording voltage changes in the other, follower cell, or (ii) measuring current flow into the follower cell. Most cell pairs of the same morphological type were coupled electrically, with coupling coefficients often greater than 0.9. Junctional resistance was typically found to be between 20 and 60 M omega and junctional conductance was between 150 and 500 nS. After application of 1-microliter pulses of dopamine (200 microM) to coupled pairs of cells, the coupling coefficient fell to approximately equal to 0.1, junctional resistance increased to 300-700 M omega, and junctional conductance decreased to 15-30 nS. Recovery of coupling took, for most cell pairs tested, 8-15 min after dopamine application. The exogenous application of 8-bromo-cyclic AMP (0.5-1 mM) also caused uncoupling of horizontal cell pairs; however, neither isoprenaline nor L-glutamate altered coupling significantly.
Polyclonal antibodies to phospholipid/Ca2+-dependent protein kinase and immunocytochemical localization of the enzyme in rat brain.
Authors: Girard P R , Mazzei G J , Wood J G , Kuo J F
Published: May 2006

Antisera against phospholipid/Ca2+-dependent protein kinase (protein kinase C) were raised in rabbits. Immunospecificity of the polyclonal antibodies, as determined by immunoblot and ELISA, was shown by their reactivity to the enzyme but not to other protein kinases or any of many other proteins tested. Immunocytochemical localization of the kinase in rat brains revealed that although the enzyme was distributed broadly in different brain regions, it was highly restricted to the periphery of the nucleus of neurons in cerebral cortex and to axons and cells strongly resembling oligodendroglia in white-matter regions. Initial electron microscopy of cerebral cortex revealed that the enzyme was highly concentrated in the presynaptic terminals, and only rarely were labeled postsynaptic specialization elements seen. It is suggested that the discrete localization of the enzyme, which is distinct from that of the calmodulin/Ca2+-dependent system, may be related to certain biological and functional aspects of brain that are regulated by Ca2+ at the level of protein phosphorylation.
Oscillatory singular integrals and harmonic analysis on nilpotent groups
Authors: Ricci F. , Stein E. M.
Published: May 2006

Several related classes of operators on nilpotent Lie groups are considered. These operators involve the following features: (
i
) oscillatory factors that are exponentials of imaginary polynomials, (
ii
) convolutions with singular kernels supported on lower-dimensional submanifolds, (
iii
) validity in the general context not requiring the existence of dilations that are automorphisms.
Development of a chemically defined serum- and protein-free medium for growth of human peripheral lymphocytes.
Authors: Shive W , Pinkerton F , Humphreys J , Johnson M M , Hamilton W G , Matthews K S
Published: May 2006

A chemically defined, protein-free medium (designated CFBI 1000, where CFBI = Clayton Foundation Biochemical Institute) that supports human peripheral lymphocyte proliferation has been developed. This medium allows exploration of individual metabolic differences by varying the medium composition as well as providing a base to explore further the mechanisms of lymphocyte activation in a system initially free of added macromolecular species other than mitogen. The peripheral blood lymphocyte is an ideal system for metabolic studies because it is easily obtained, is a primary resting cell that can be activated to proliferate, and presumably reflects both the genetic makeup and biochemical environmental history of the individual at the time the cells were formed. Examination of the role of various factors in lymphocyte activation and subsequent events may be simplified by the utilization of a medium that is protein-free and chemically defined. The CFBI 1000 medium supports the growth response of human peripheral lymphocytes to mitogen as measured by [3H]thymidine incorporation to an extent comparable to other media used widely in assessment of lymphocyte proliferation.
Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells.
Authors: Yu S F , von Rüden T , Kantoff P W , Garber C , Seiberg M , Rüther U , Anderson W F , Wagner E F , Gilboa E
Published: May 2006

A retrovirus-derived vector called self-inactivating (SIN) vector was designed for the transduction of whole genes into mammalian cells. SIN vectors contain a deletion of 299 base pairs in the 3' long terminal repeat (LTR), which includes sequences encoding the enhancer and promoter functions. When viruses derived from such vectors were used to infect NIH 3T3 cells, the deletion was transferred to the 5' LTR, resulting in the transcriptional inactivation of the provirus in the infected cell. Introduction of a hybrid gene (human metallothionein-promoted c-fos) into cells via a SIN vector was not associated with rearrangements and led to the formation of an authentic mRNA transcript, which in some cases was induced by cadmium. SIN vectors should be particularly useful in gene transfer experiments designed to study the regulated expression of genes in mammalian cells. Absence of enhancer and promoter sequences in both LTRs of the integrated provirus should also minimize the possibility of activating cellular oncogenes and may provide a safer alternative to be used in human gene therapy.
Photoreactivation rescue and hypermutability of ultraviolet-irradiated excisionless
Drosophila melanogaster
larvae
Authors: Ryo Haruko , Kondo Sohei
Published: May 2006

There is accumulating evidence suggesting that expression of genes for repair of UV damage to DNA in mammals and fish is regulated developmentally. Therefore, the activity of excision repair and photoreactivation
in vivo
in young larvae of
Drosophila melanogaster
was examined in a strain carrying the mutation
mus201
that was unable to carry out excision repair. The photoreactivation activity in first-instar larvae was so high that UV-induced lethality in excision-less larvae was almost completely rescued by posttreatment with fluorescent light. Excision repair activity in first-instar repair-proficient larvae was so high that UV irradiation was scarcely able to produce somatic eye-color mutations. In contrast, excisionless larvae showed a high incidence of somatic eye-color mutation after UV-irradiation, and this incidence was reduced to the spontaneous level by posttreatment with fluorescent light. Incorporation of a postreplication repair-defective mutation into the excisionless strain decreased the incidence of UV-induced somatic mutations by a factor of 3. The analogous repair dependence of UV mutagenesis in
Drosophila
and
Escherichia coli
is discussed. It is proposed that UV-induced somatic mutations in excisionless
Drosophila
larvae are caused primarily by pyrimidine dimers and that a constitutive, error-prone pathway for filling daughter-strand gaps opposite dimers is, at least partly, responsible for the fixation of mutations.
Formal proof that different-size Lyt-2 polypeptides arise from differential splicing and post-transcriptional regulation.
Authors: Tagawa M , Nakauchi H , Herzenberg L A , Nolan G P
Published: May 2006

We recently isolated the gene and a cDNA clone for the mouse T-cell surface antigen Lyt-2 and showed that Lyt-2 is homologous to the human Leu-2 (T8) antigen and that the gene encoding it is a member of the immunoglobulin gene superfamily. By screening a mouse thymus cDNA library with the Lyt-2 cDNA clone, we isolated two classes of cDNA clones, alpha and alpha', which differ by 31 base pairs. Comparison of the alpha cDNA with genomic sequence data indicates that there are five exons encoding Lyt-2: a fused leader/immunoglobulin variable region-like exon, a spacer region exon, a transmembrane exon, and two cytoplasmic exons. The alpha' cDNA clones lack the first of the two cytoplasmic exons and have a direct splice from the donor splice site of the transmembrane exon to the acceptor of the second cytoplasmic exon. This splice changes the reading frame for the second cytoplasmic exon, causing a stop codon shortly after the splice so that the alpha' cDNA clone codes for a peptide 25 residues shorter than the alpha cDNA-encoded peptide. We have constructed expression vectors with alpha and alpha' cDNAs and have shown that L-cell transfectants of these produce Lyt-2 polypeptides of the predicted sizes and that these associate as homodimers on the cell membranes. We found the two species of mRNA corresponding to alpha and alpha' cDNAs at equal levels in thymus RNA by using S1 nuclease analysis. Although lymph node T cells have only the alpha form of Lyt-2 protein, S1 nuclease analysis shows that lymph nodes have about 20% alpha' mRNA relative to alpha. Thus, Lyt-2 is regulated at RNA processing, translational, and/or post-translational steps.
Voltage- and time-dependent effects of phencyclidines on the endplate current arise from open and closed channel blockade.
Authors: Aguayo L G , Witkop B , Albuquerque E X
Published: May 2006

The actions of phencyclidine [1-(1-phenylcyclohexyl)piperidine, PCP] and its morpholine analog [1-(1-phenylcyclohexyl)morpholine, PCM] on ionic currents of nicotinic acetylcholine receptors were studied at the neuromuscular junction of frog skeletal muscle and on embryonic rat muscle cells in tissue culture. PCP and PCM reduced the peak amplitude and the decay time constant of the endplate current (EPC). PCP produced a voltage-dependent curvature and a time-dependent hysteresis loop at negative potentials (at potentials from -50 to -150 mV). In contrast, PCM caused a depression of EPC peak amplitude, but the current-voltage relationship (+60 to -150 mV) remained linear. When PCP-modified EPCs were elicited in trains at hyperpolarized potentials the amplitudes of successive events were progressively decreased and the magnitude of the decrease was dependent on the level of hyperpolarization. At positive potentials the process was reversed; the amplitude increased with successive stimulations. The EPC decayed exponentially in the presence of PCP and PCM, with a shortened time constant of decay that was less dependent on membrane potential than control. PCP and PCM caused only a 20% decrease of the amplitude of the iontophoretically evoked acetylcholine potential, which was significantly different from that induced by the desensitizing alkaloid perhydrohistrionicotoxin. Both PCP and PCM reduced by 50% the mean channel open time obtained from rat myoballs, giving a potency ratio for PCP to PCM of 2.5. This relative potency was correlated with that obtained for the reduction in the decay time constant of the EPC (ratio = 2.2). The effects of PCP on the peak amplitude of the EPC seem to be related to a conformational change of the acetylcholine receptor occurring before channel activation and not to a receptor desensitization.
Genetic uniformity in two populations of Drosophila melanogaster as revealed by filter hybridization of four-nucleotide-recognizing restriction enzyme digests.
Authors: Kreitman M , Aguadé M
Published: May 2006

A filter hybridization method is described for identifying restriction-site and insertion/deletion variation by using restriction enzymes that recognize four-nucleotide sequences and denaturing polyacrylamide gels for separating fragments. Eighty-seven lines of Drosophila melanogaster representing two natural populations were surveyed over a 2.7-kilobase region encompassing the alcohol dehydrogenase locus. Fifty distinct haplotypes were identified from 17 restriction-site and 11 insertion/deletion polymorphisms and from one allozyme polymorphism. There was no evidence for genetic differentiation between an East-Coast and a West-Coast (North American) sample. This technique has widespread applications in screening for DNA polymorphism.
Transient replication of bovine papilloma virus type 1 plasmids: cis and trans requirements.
Authors: Lusky M , Botchan M R
Published: May 2006

A transient assay has been used to study bovine papilloma virus type 1 (BPV-1) replication. We show that BPV-1 early replication occurs faster than cellular DNA synthesis. Initial replication events are dependent on a gene product(s), encoded by the BPV-1 E1 open reading frame. Mutational analysis of the viral upstream regulatory region shows the requirement of two domains in cis for replication. Domain one, located outside of the viral 69% transforming fragment, is an enhancer-like activity and can be replaced by other known viral enhancers. Domain two lies within sequences previously defined as plasmid maintenance sequence 1. The apparent requirement for a proximal enhancer function for replication may explain why certain BPV-1 constructions, when linked to bacterial plasmid sequences, can be maintained extrachromosomally while others cannot.
Exon-Alu recombination deletes 5 kilobases from the low density lipoprotein receptor gene, producing a null phenotype in familial hypercholesterolemia.
Authors: Lehrman M A , Russell D W , Goldstein J L , Brown M S
Published: May 2006

Among patients with familial hypercholesterolemia, half of the mutant alleles at the low density lipoprotein (LDL) receptor locus produce no immunologically detectable protein. To determine the molecular basis for one such null allele, we have cloned an abnormally short restriction fragment from the genomic DNA of one patient. The DNA sequence revealed a 5-kilobase deletion that joins a coding sequence in exon 13 to an Alu repetitive element in intron 15. The deletion joint is flanked by two inverted repeats that could potentially form a double stem-loop structure that might have predisposed to this deletion. A similar double stem-loop structure can be drawn for a previously described deletion in the LDL receptor gene and for a deletion in the beta-globin gene cluster. We speculate that such double stem-loop structures might contribute to the formation of large deletions in the human genome.
Identification of a telomere-binding activity from yeast.
Authors: Berman J , Tachibana C Y , Tye B K
Published: May 2006

In yeast, the ends of the chromosomes (telomeres) terminate in repeated poly(C1-3A) sequences. We have identified a yeast activity that binds specifically to these poly(C1-3A) repeats. An agarose gel binding assay was used to detect and characterize this activity in cell extracts using both cloned telomere DNA and yeast genomic DNA as substrates. The activity appears to bind specifically to poly(C1-3A) sequences, despite their different primary sequences, yet does not bind specifically to telomeric repeats, such as poly(C4A2), poly(C4A4), and poly (C1-8T) from other lower eukaryotes.
Complete amino acid sequence of chicken cartilage link protein deduced from cDNA clones.
Authors: Deák F , Kiss I , Sparks K J , Argraves W S , Hampikian G , Goetinck P F
Published: May 2006

cDNA clones coding for chicken cartilage link protein were isolated and sequenced. The DNA sequence for the entire core polypeptide of the mature link protein and the predicted signal peptide consists of 1065 nucleotides. The deduced primary translation product (355 amino acids) has a molecular mass of 40.7 kDa; the calculated molecular mass of the mature link protein core polypeptide (340 amino acids) is 39.06 kDa. The DNA sequence contains two tandemly arranged repeat sequences that may code for repeated functional domains of link protein involved in binding to hyaluronic acid. The mRNAs for chicken link protein are 6.0, 5.8, and 3.0 kilobase pairs, and the difference between the sizes of the RNA species lies in the 3' untranslated region.
Expression of different-sized placental alkaline phosphatase mRNAs in placenta and choriocarcinoma cells.
Authors: Ovitt C E , Strauss A W , Alpers D H , Chou J Y , Boime I
Published: May 2006

The expression of human placental-type alkaline phosphatase (ALPase) in the placenta and in three choriocarcinoma cell lines was examined by translation in vitro and RNA blot analysis using a cDNA for placental ALPase. Placental RNA directed the synthesis of two polypeptides that could be immunoprecipitated with antiserum to placental ALPase. Translation of RNA from the choriocarcinoma cell lines, with or without sodium butyrate treatment, yielded a single immunoprecipitable product of molecular weight intermediate between those of the products from the placenta mRNA. Two cDNA clones for placental ALPase were isolated by antibody screening of a placental cDNA library constructed in lambda gt11. The overlapping cDNAs include 462 nucleotides of coding sequence. RNA blot analysis has confirmed that induction of placental-type ALPase levels during placental development is accompanied by an increase in steady-state placental ALPase mRNA concentrations. Examination of the mRNAs revealed a placental ALPase mRNA of 3.0 kilobases (kb) and a distinct choriocarcinoma placental-type ALPase mRNA of 2.6 kb, implying that transformation of normal to malignant trophoblast is associated with the expression of a distinct placental-type ALPase gene transcript and its protein product.