bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunohistochemical evidence for the coexistence of histidine decarboxylase-like and glutamate decarboxylase-like immunoreactivities in nerve cells of the magnocellular nucleus of the posterior hypothalamus of rats.
Authors: Takeda N , Inagaki S , Shiosaka S , Taguchi Y , Oertel W H , Tohyama M , Watanabe T , Wada H
Published: May 2006

Immunohistochemical staining of alternate consecutive sections revealed numerous histidine decarboxylase (L-histidine carboxy-lyase, EC 4.1.1.22)-like immunoreactive neurons that also contained glutamate decarboxylase (L-glutamate 1-carboxy-lyase, EC 4.1.1.15)-like immunoreactive structures in the tuberal magnocellular nucleus, the caudal magnocellular nucleus, and the postmammillary caudal magnocellular nucleus of the posterior hypothalamus of rats. Furthermore, in immunohistochemical double-staining procedures, almost all neurons in the magnocellular nuclei had both histidine decarboxylase-like and glutamate decarboxylase-like immunoreactivities. These results suggest the coexistence of histamine and gamma-aminobutyric acid in single neurons in these nuclei.
Gamma-crystallin family of the mouse lens: structural and evolutionary relationships.
Authors: Breitman M L , Lok S , Wistow G , Piatigorsky J , Tréton J A , Gold R J , Tsui L C
Published: May 2006

The heterogeneity inherent among gamma-crystallins of the mouse lens was investigated by sequence analysis of three gamma-crystallin-specific cDNAs. Comparison of the nucleotide sequence of these cDNAs and one previously reported by us revealed that the four gamma-cDNAs share 80-90% homology in nucleotide sequence. The entire 3' half of the coding region shows more variability than the 5' half, whereas the greatest variability is observed in the 3' untranslated region where numerous base substitutions, deletions, and insertions seem to have occurred. Alignment of the amino acid sequences of the four mouse gamma-crystallins according to the known four structural motifs of the major calf gamma-crystallin, gamma-II, suggests that all four mouse polypeptides are structurally very similar to calf gamma-II. However, most of the mouse polypeptides differ from gamma-II by the absence of one amino acid residue, resulting in a shorter connecting peptide between the two globular domains of the protein. Primary sequence alignment also revealed that the four mouse gamma-crystallins are most divergent in the third structural motif of the polypeptide. The significance of these differences in terms of the structure and function of the gamma-crystallins in the mouse lens is discussed.
alpha-Amanitin-insensitive transcription of mouse beta major-globin 5'-flanking and structural gene sequences correlates with mRNA expression.
Authors: Carlson D P , Ross J
Published: May 2006

A small proportion of the RNAs of mouse reticulocytes consists of beta major-globin mRNA sequences linked to sequences transcribed from the 5'-flanking region of the beta major-globin gene. These upstream RNAs are polyadenylylated and contain 700-800 nucleotides, and their 5' regions are heterogeneous. RNAs with similar or identical 5' regions are transcribed in cell-free extracts from a circular mouse beta major-globin gene template. Synthesis of most of the upstream RNAs in vitro is not inhibited by low levels (1 microgram/ml) of alpha-amanitin, indicating that they are transcribed by an enzyme(s) different from RNA polymerase II. During culture of mouse erythroleukemia cells with dimethyl sulfoxide, globin mRNA and upstream RNAs accumulate with similar kinetics. In contrast, upstream RNAs are not detected in hemin-treated cells.
Angiotensin II rapidly increases phosphatidate-phosphoinositide synthesis and phosphoinositide hydrolysis and mobilizes intracellular calcium in cultured arterial muscle cells.
Authors: Smith J B , Smith L , Brown E R , Barnes D , Sabir M A , Davis J S , Farese R V
Published: May 2006

Smooth muscle cells were cultured from rat thoracic aorta and labeled to a stable specific activity with 45Ca2+, myo-[2-3H]inositol, or 32Pi. The efflux of 45Ca2+ was monitored over 10-sec intervals. Angiotensin II (AII) increased the amount of 45Ca2+ lost by 5-fold in the first 10-sec interval after the addition of AII and by 10-fold in the second 10-sec interval. AII-stimulated 45Ca2+ release was blocked by the angiotensin antagonist [1-sarcosine, 8-leucine]AII and by La3+. The removal of external Ca2+ had no effect on AII-stimulated 45Ca2+ release. Depolarization with high external K+ only slightly increased 45Ca2+ efflux and had no effect on AII-induced 45Ca2+ release. AII had no effect on the initial rate of 45Ca2+ influx. These results indicate that the rapid 45Ca2+ efflux evoked by AII is probably due to the release of 45Ca2+ sequestered intracellularly rather than to an increase in the Ca2+ permeability of the plasma membrane. AII provoked rapid increases in the levels of phosphatidic acid and phosphoinositides in the cells. These increases in phospholipids were associated with increases in phospholipase C-generated inositol phosphates (tri-, di-, and mono-). It appears that AII simultaneously increases phosphoinositide hydrolysis and synthesis in vascular smooth muscle, and both phospholipid effects may contribute to inositol triphosphate generation, which was sufficiently rapid to have a role in intracellular Ca2+ mobilization.
Cytological mapping of the human glucose-6-phosphate dehydrogenase gene distal to the fragile-X site suggests a high rate of meiotic recombination across this site.
Authors: Szabo P , Purrello M , Rocchi M , Archidiacono N , Alhadeff B , Filippi G , Toniolo D , Martini G , Luzzatto L , Siniscalco M
Published: May 2006

The human gene for glucose-6-phosphate dehydrogenase (G6PD) has been subregionally mapped to band Xq28 by segregation analysis in rodent-human somatic cell hybrids [Pai, G. S., Sprinkel, J. A., Do, T. T., Mareni, C. E. & Migeon, B. R. (1980) Proc. Natl. Acad. Sci. USA 77, 2810-2813]. We have previously reported a common type of X-linked mental retardation associated with an inducible fragile site at Xq27-Xq28 segregates in a close linkage relationship with a G6PD variant, but the relative position of G6PD with respect to the fragile site has not yet been established. This fragile-X syndrome has been shown to be closely linked also to a Taq I restriction fragment length polymorphism detected by a cDNA probe for factor IX, and the latter locus has been mapped to the subtelomeric region Xq26-Xq28 [Camerino, G., Mattei, M. G., Mattei, G. F., Jaye, B. & Mandel, J. L. (1983) Nature (London) 306, 701-704]. The in situ hybridization studies reported here provide strong evidence that G6PD is located on the Xq telomeric fragment distal to the fragile site. These observations and the well-established knowledge that the genes for Deutan and Protan colorblindness are closely linked to G6PD, but segregate independently of factor IX deficiency, suggest that the fragile site associated with this type of X-linked mental retardation occurs in a region prone to high frequency of meiotic recombination.
Aminomalonic acid: identification in Escherichia coli and atherosclerotic plaque.
Authors: Van Buskirk J J , Kirsch W M , Kleyer D L , Barkley R M , Koch T H
Published: May 2006

Aminomalonic acid (Ama) has been isolated from proteins of Escherichia coli and human atherosclerotic plaque. The presence of Ama has important biological implications because the malonic acid moiety potentially imparts calcium binding properties to protein. Ama was obtained by anaerobic alkaline hydrolysis and identified by chromatographic behavior, quantitative acid-mediated decarboxylation to glycine, and unambiguous gas chromatographic/mass spectral detection. The chromatographic, chemical, and mass spectral properties of naturally occurring Ama were identical to those of the synthetic compound. Amino acid analysis and GC/mass spectrometry also revealed the presence of beta-carboxyaspartic acid and gamma-carboxyglutamic acid in the base hydrolysate of human atherosclerotic plaque. The ratio of Ama to beta-carboxyaspartic acid to gamma-carboxyglutamic acid was 20:1:10, and the quantity of Ama per 1,000 glycine residues was 0.2. Ama is a relatively unstable, minor amino acid in complex structures such as bacteria or tissues. This may explain why it has escaped detection previously, despite intensive investigation.
Altered temporal expression of DNA repair in hypermutable Bloom's syndrome cells.
Authors: Gupta P K , Sirover M A
Published: May 2006

The temporal regulation of DNA repair during synchronous cell proliferation was examined in normal human skin fibroblasts and in Bloom's syndrome skin fibroblasts. Normal human cells regulated DNA repair in a defined temporal sequence prior to the induction of DNA replication. Nucleotide-excision repair was stimulated prior to the induction of base-excision repair, which itself was increased prior to the induction of DNA replication. This temporal sequence was observed (i) by quantitation of the induction of the base-excision repair enzyme uracil DNA glycosylase during cell proliferation in the absence of cellular insult and (ii) by quantitation of nucleotide-excision repair after UV irradiation or base-excision repair after exposure to methylmethane sulfonate. In contrast, Bloom's syndrome cells were characterized by specific alterations in this temporal sequence of gene regulation, such that DNA repair was not enhanced prior to the induction of DNA replication. Nucleotide-excision repair, base-excision repair, and the uracil DNA glycosylase were induced in a temporal sequence identical to that observed for DNA polymerase and for DNA replication. The inability of Bloom's syndrome cells to enhance DNA repair prior to DNA replication suggests that miscoding lesions remain in DNA and are replicated during cell proliferation.
Insulin-induced reactivation of an inactive herpes simplex thymidine kinase gene.
Authors: Clough D W , Morse B S , Kucherlapati R S , Davidson R L
Published: May 2006

A line of mouse cells transformed with ultraviolet-irradiated herpes simplex virus type 1 and containing a methylated and inactive viral thymidine kinase (TK) gene was treated with insulin in an attempt to induce expression of the inactive gene. Insulin was found to be capable of inducing the inactive TK gene in these cells. The induction of the TK+ phenotype was dose dependent (from 1-100 micrograms of insulin per ml), and the TK activity induced was shown to be of viral origin. Analysis of the methylation pattern of the viral TK gene by using the methylation-sensitive restriction endonucleases Sma I, Hpa II, and Hha I revealed that the active viral TK gene in the parental transformed cells was hypomethylated, whereas the inactive TK gene in the uninduced TK- cells was methylated. The active TK gene in three insulin-induced TK+ lines also was methylated, but the methylation patterns in the insulin-induced lines all were different from the uninduced TK- line. These data suggest that extensive hypomethylation of the inactive TK gene is not required for insulin induction. Four other transformed lines containing an inactive viral TK gene were tested for insulin inducibility, but insulin was unable to induce expression of the TK gene in any of the other lines. Thus, insulin inducibility does not seem to be a function of the viral TK gene itself. These results suggest that insulin inducibility of the viral TK gene may be a reflection of the region of the host genome into which the TK gene was integrated.
Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous.
Authors: Bardwell J C , Craig E A
Published: May 2006

The Escherichia coli dnaK gene is homologous to the major heat shock-induced gene in Drosophila (Hsp70). The primary DNA sequence of the entire protein-coding region of the dnaK gene was determined and compared with that of the Hsp70 gene of Drosophila. The two sequences are homologous; the dnaK gene could encode a 69,121-Da polypeptide, 48% identical to the hsp70 protein of Drosophila. The homology between the Hsp70 gene of Drosophila and the E. coli dnaK gene illustrates the remarkable conservation of the heat shock genes in evolution. In contrast to Drosophila and Saccharomyces cerevisiae, both of which contain multigene families related to the Hsp70 gene, hybridization analyses indicate that E. coli contains only a single Hsp70-related gene, dnaK. Hybridization between the DNA of an archaebacterium Methanosarcina barkeri and the Hsp70 genes of Drosophila, Saccharomyces, and E. coli has been detected, suggesting the existence of Hsp70-related genes in the three "primary kingdoms": eukaryotes, eubacteria, and archaebacteria.
Characterization of a membrane antigen from rabbit testis and sperm isolated by using monoclonal antibodies and effect of its antiserum on fertility.
Authors: Naz R K , Rosenblum B B , Menge A C
Published: May 2006

An antigen was isolated from deoxycholate-solubilized rabbit testis and sperm by using an immunosorbent column containing IgG from a monoclonal antibody (8C10.5) that inhibits fertility. Elution was by stepwise increases in pH (8.0, 10.0, and 11.4), with the pH 11.4 fraction after recycling through the column showing a single band at 63 kilodaltons in slab NaDodSO4/PAGE with a silver stain. The antigen molecule was composed of two subunits, which on two-dimensional PAGE showed many spots within the same molecular size range (50-70 kilodaltons) but differing in charge. The antigen isolated either from testis or sperm showed mainly the same spots. The antigen is periodic acid/Schiff positive and contained 21% carbohydrate. An asialo-derivative of the antigen did not change its characteristics on NaDodSO4/PAGE. This glycoprotein resolved into two types of polypeptides, those binding and those not binding to a lens culinaris lectin column; some of the polypeptides appeared common to both fractions. Murine antiserum against the antigen neither agglutinated nor immobilized rabbit sperm but in immunofluorescence reacted with the plasma membrane of viable rabbit sperm as well as with murine and human sperm. Fertilization of female rabbits inseminated with treated sperm was not affected, but, by 9 days, fertility was significantly reduced (21% of controls). The postfertilization antifertility effect was not due to parthenogenic activation or to polyspermy. The antiserum reacted with one specific band in the one- and two-dimensional gel electrophoretic transfer blot procedure and was unaffected by absorption with different somatic tissues.
99mTc-labeled nucleotides as tumor-seeking radiodiagnostic agents.
Authors: Elmaleh D R , Zamecnik P C , Castronovo F P , Strauss H W , Rapaport E
Published: May 2006

Several lines of human tumor cells in monolayer and soft agar cultures allow permeation of low levels of adenine nucleotides through their plasma membranes, while, in general, untransformed cells do not incorporate adenine nucleotides into their cellular pools without prior degradation of the nucleotides to adenosine. This study determined the uptake of 99mTc-radiolabeled chelated forms of adenine nucleotides, 99mTc-Ap4A (diadenosine 5',5"',P1,P4-tetraphosphate) and 99mTc-ATP chelates as radiodiagnostic agents suitable for the in vivo detection of tumors by radionuclide imaging. Biodistribution studies revealed that Ap4A accumulated preferentially in RT-24 tumors implanted in rats and that V2 carcinoma implanted in rabbits could be readily visualized by in vivo imaging. The biodistribution at various time points showed increased tumor-to-muscle ratios after 99mTc-Ap4A or 99mTc-ATP injections when compared with a nonspecific marker of the extracellular fluid space, 99mTc-labeled diethylenetriaminepentaacetic acid and with an agent known to localize in some tumors, 67Ga-labeled citrate. Studies of ectoenzymatic activities of virus-transformed animal cells and their untransformed counterparts in monolayer cultures showed marked decreases in the ectoenzymatic activities that degrade Ap4A in the transformed cells. Incorporation of en bloc [3H, 32P]Ap4A into cellular acid-soluble nucleotide pools of certain transformed cells was observed. Normal untransformed cells incorporated the radioactive label only by prior degradation to [3H]adenosine and 32Pi.
Regulation of protein metabolism: Coupling of photosynthetic electron transport to
in vivo
degradation of the rapidly metabolized 32-kilodalton protein of the chloroplast membranes
Authors: Mattoo Autar K. , Hoffman-Falk Hedda , Marder Jonathan B. , Edelman Marvin
Published: May 2006

In
Spirodela oligorrhiza
, mature chloroplasts copiously synthesize and degrade a 32-kilodalton membrane protein. The rates of synthesis and degradation are controlled by light intensity, the protein being unstable in the light and stable in the dark. Light-driven synthesis, but not degradation, is dependent on ATP. Degradation is blocked by herbicides inhibiting photosystem II electron transport, such as diuron and atrazine. Thus, both anabolism and catabolism of the 32-kilodalton protein are photoregulated, with degradation coupled to electron transport rather than phosphorylation.
Distribution of prothymosin alpha in rat tissues.
Authors: Haritos A A , Tsolas O , Horecker B L
Published: May 2006

A radioimmunoassay, using a rabbit antiserum directed against thymosin alpha 1, was employed to detect the presence of crossreacting peptides in rat tissues. Highest concentrations were present in thymus, but thymosin alpha 1 cross-reacting material was also detected in brain, liver, kidney, lung, and spleen, in amounts ranging from 15% to 65% of the quantities found in thymus. In each case, the major immunoreactive peptide, after extraction and purification by a procedure that avoids proteolytic modification, was identified as prothymosin alpha, a peptide containing approximately equal to 112 amino acid residues. Prothymosin alpha is believed to be the endogenous peptide from which thymosin alpha 1 and other fragments are formed by proteolytic modification during the preparation of thymosin fraction 5. No peptides corresponding in size and chromatographic behavior to thymosin alpha 1 were detected with the extraction procedure employed.
Virus-plasmid interactions: mutants of bacteriophage T3 that abortively infect plasmid F-containing (F+) strains of Escherichia coli.
Authors: Molineux I J , Spence J L
Published: May 2006

Bacteriophage T7 and many closely related phages abortively infect plasmid F-containing (F+) strains of Escherichia coli. However phage T3, which is also closely related to T7, grows normally in F+ hosts. Mutants of phage T3 that, like T7, are subject to F-mediated restriction have been isolated. These T3 mutants lack or are defective in one or both of two genes that are nonessential for phage growth in F-, wild-type strains. Our results show that the products of phage T3 gene 1.1 or 1.2, or both, are essential for growth and suggest that the comparable phage T7 genes are naturally defective in their ability to counteract the inhibitory effects of F-encoded proteins.
Mutational specificity of depurination.
Authors: Kunkel T A
Published: May 2006

The mutagenic consequences of damage to DNA produced by low pH and high temperature have been determined in a forward mutational system capable of detecting all classes of mutagenic events. When damaged single-stranded DNA from bacteriophage M13mp2 is used to transfect competent Escherichia coli cells, a 15-fold increase in mutation frequency, measured as loss of alpha-complementation by the lac DNA in the phage, is observed compared with an untreated DNA control transfection. The enhanced mutagenicity is largely dependent on induction of the error-prone SOS response and is proportional to the number of lethal hits introduced into the DNA. The effect is abolished by treatment of the damaged DNA before transfection with either apurinic/apyrimidinic endonuclease or alkali. Based on these observations and the rate constants for formation of the known heat/acid-produced lesions in DNA, it is concluded that the majority of the induced mutagenesis results from apurinic sites. DNA sequence analysis of 87 spontaneous and 124 induced mutants indicates that the major effect is on single base-substitution mutagenesis with a small increase in (deletion) frame-shift frequency. Approximately 80% of the base-substitution mutations occur at purine positions in the viral strand, consistent with depurination as the predominant premutagenic lesion. The preference of guanine over adenine sites mutated is consistent with the preference for depurination of guanine over adenine. Transversions are observed for 57 of 79 (72%) induced base substitutions, with a strong preference for insertion of adenine residues opposite the putative apurinic site. These data in a forward mutational system provide insight into the mechanisms used by a cell to replicate DNA containing noncoding lesions.
Growth hormone-releasing factor regulates growth hormone mRNA in primary cultures of rat pituitary cells.
Authors: Gick G G , Zeytin F N , Brazeau P , Ling N C , Esch F S , Bancroft C
Published: May 2006

A peptide with high intrinsic activity for specifically stimulating the secretion of immunoreactive growth hormone (GH; somatotropin) has been characterized and reproduced by total synthesis. This peptide, human pancreatic growth hormone-releasing factor, 44-amino-acid form (hpGRF1-44-NH2), was isolated from a tumor localized in the pancreas of a patient with acromegaly. We report here the effect of this growth hormone-releasing factor (GRF) on GH release and the GH mRNA levels in monolayer cultures of rat pituitary. The cytoplasmic dot hybridization technique was used to examine the effect of GRF on GH mRNA levels. Incubation of rat pituitary cultures with GRF for 72 hr resulted in a 2- to 2.5-fold increase in GH mRNA levels, and the maximal levels of stimulation were achieved at GRF concentrations as low as 1 fM. GRF did not stimulate prolactin release, nor did it affect specific prolactin mRNA levels in the pituitary cultures. We conclude that GRF is a potent and specific GH secretagogue that also affects specifically GH mRNA levels in normal pituitary cells.
Mechanism of T-cell lymphomagenesis: transformation of growth-factor-dependent T-lymphoblastoma cells to growth-factor-independent T-lymphoma cells.
Authors: Haas M , Altman A , Rothenberg E , Bogart M H , Jones O W
Published: May 2006

In a previous paper we described the induction by x-irradiation or radiation-induced leukemia virus-inoculation of two classes of lymphoid T-cell neoplasms: The first class, designated T-cell lymphoblastoma (TCLB), consists of growth-factor-dependent eudiploid cells that home to the spleen and give rise to splenic tumors on injection into syngeneic mice; the second class, designated T-cell lymphoma (TCL), consists of growth-factor-independent aneuploid or pseudodiploid cells that give rise to local tumors at the site of subcutaneous injection. This paper describes the generation of a family of growth-factor-independent aneuploid or pseudodiploid TCL cells after the injection into the thymus of growth-factor-dependent diploid TCLB cells. In contrast to the donor TCLB cells, the resulting TCL cells could be cloned in semisolid medium, produced local tumors at the site of subcutaneous injection, and proliferated in a growth-factor-independent fashion in vitro. The induced growth-factor-independent TCL cells were chromosomally and phenotypically unstable and continued to evolve both in vivo and in vitro. After propagation in the thymus, the cells often showed stable translocations in addition to the evolving aneuploidy. We propose that the chromosome abnormalities induced during the proliferation of growth-factor-dependent TCLB cells in the thymus constitute a general mechanism by which neoplastic cells progress from growth-factor dependency to independency.
Membrane proteins unique to vertebrate olfactory cilia: candidates for sensory receptor molecules.
Authors: Chen Z , Lancet D
Published: May 2006

In search for olfactory receptor molecules, we carried out comprehensive electrophoretic mapping of membrane proteins in the cilia of frog olfactory epithelium. Seven polypeptides, extracted from isolated cilia by nonionic detergent, were unique to the sensory organelles, compared to nonsensory (respiratory) counterparts. Olfactory cilia contained 3-10 times more membrane-associated protein as compared to respiratory cilia, in agreement with reported densities of freeze-fracture intramembranous particles. Four of the olfactory polypeptides were major constituents of the ciliary membrane, each amounting to greater than 10% of its total protein. Three major and one minor specific polypeptide were glycosylated, whereas membranes of nonsensory cilia were practically devoid of glycoproteins. A clear difference in surface composition was also shown by microscopic visualization of fluoresceinated lectin bound to intact isolated cilia. Two of the olfactory glycoproteins displayed pronounced heterogeneity of apparent molecular weight, which could partly be due to protein sequence diversity, as expected for odorant receptor molecules. The recently described inhibition of odorant-evoked sensory potentials by the lectin concanavalin A is consistent with the hypothesis that one or more of the specific glycoproteins described here plays a role in olfactory reception.
Mechanoelectrical transducer has discrete conductances in the chick vestibular hair cell.
Authors: Ohmori H
Published: May 2006

Properties of mechanoelectrical transduction were studied at the single-cell level by applying a whole-cell recording variation of the patch-clamp technique to dissociated vestibular hair cells of chicks. The hair bundle was directly stimulated by a glass rod, and transduction currents were recorded from the cell body. After a triangular movement of the stimulating probe, the transduction current was generated stepwise between discrete levels of amplitude. The minimum step amplitude was -1.8 pA at -27 mV in Na-containing normal saline.
Enzymatic techniques for the isolation of random single-base substitutions in vitro at high frequency.
Authors: Abarzúa P , Marians K J
Published: May 2006

A general and efficient method has been developed to generate large numbers of single-base substitution mutations simply and rapidly. A unique f1 phage recombinant DNA cloning vector is described, which contains the phi X174 origin of viral strand DNA synthesis and allows one to direct mutagenesis to any specific segment of DNA. Gapped circular DNA is constructed by annealing viral single-stranded circular DNA [ss(c) DNA] with a mixture of linear duplex DNAs that have had their 3'-OH termini processively digested with Escherichia coli exonuclease III under conditions in which the resulting, newly generated 3'-OH termini present in the various hybrid molecules span the region of interest. Base changes are induced by misincorporation of an alpha-thiodeoxynucleoside triphosphate analog onto this primer-template, followed by DNA repair synthesis. The asymmetric segregation of mutants from wild-type sequences is accomplished by double-stranded replicative form DNA----ss(c) DNA synthesis in vitro, initiated from the phi X174 viral strand origin sequence present on the vector DNA. Mutated ss(c) DNA is screened by the dideoxy chain termination method. In one mutagenesis experiment, 21 independent single-base substitutions were isolated in a 72-nucleotide-long target region. DNA sequence analysis showed that all possible base transversions and transitions were represented.
Malignant growth in the normal host after variant selection in vitro with cytolytic T-cell lines.
Authors: Wortzel R D , Urban J L , Schreiber H
Published: May 2006

Using variant selection in vitro, we have explored the changes necessary to convert potentially malignant cells into progressively growing cancer cells. As a model, we studied the murine ultraviolet light (UV)-induced tumor 1591-RE, which routinely regresses in the normal syngeneic host. The antigenicity of this tumor is specified by multiple independent tumor-specific antigens, named A, B, C, and D. Cells of this tumor were exposed in vitro to cytolytic T-cell lines specific for these separate antigens. Tumor variants were isolated that displayed selective antigenic losses. By challenging normal syngeneic mice with the variants, we could determine the significance of each antigen in tumor rejection--i.e., a switch from a regressor to a progressor phenotype upon selective loss of certain antigens. We found that variants which lost the A and C antigens grew progressively in normal mice, whereas variants which had lost the B antigen were still rejected like the parental tumor cells. Furthermore, the B and D antigens could be expressed on progressively growing variants. Thus, loss of the A and C antigens, but not the B and D antigens, was required for allowing malignant growth. Selection for loss of one T-cell-recognized antigen never selected simultaneously for loss of any of the other T-cell-recognized antigens, nor did such selections affect sensitivity to activated macrophages or natural killer cells. Therefore, our results strongly suggest that loss of defined components of a complex T-cell-recognized antigen are sufficient to allow escape from immunosurveillance.
Hepatitis B viral antigenic structure: signature analysis by monoclonal radioimmunoassays.
Authors: Wands J R , Wong M A , Shorey J , Brown R D , Marciniak R A , Isselbacher K J
Published: May 2006

An approach has been developed for the analysis of hepatitis B viral (HBV) antigenic structure that creates numerical "signatures" of HBV strains. This technique employs high-affinity IgM and IgG monoclonal antibodies (anti-HBsAg) directed toward distinct and separate determinants on hepatitis B surface antigen (HBsAg). Such antibodies have been used to develop sensitive and specific radioimmuno-assays for measurement of HBsAg-associated determinants in serum. In performing "signature" analysis separate binding curves for each monoclonal anti-HBsAg are generated by measuring immunoreactivity in serial dilutions of HBsAg-positive serum. Since the HBsAg concentration in serum is unknown, the binding profiles of groups of samples from the same "classic" HBV subtype are aligned by an iterative maximum likelihood procedure to give the numerical signature of that HBV subtype. By using this approach, HBsAg shows far more antigenic heterogeneity than previously recognized by polyvalent anti-HBsAg antibodies. Indeed, there are subgroups within the classic HBsAg subtypes. In addition, the a domain (common to all known subtypes or strains of HBV) has been shown to be multideterminant. Thus, these studies have demonstrated heretofore unrecognized differences in HBV subtypes. This approach also has broader significance for the study of subtle or major antigenic changes among other viral agents since it is not necessary to know the concentration of virus or viral protein in complex protein mixtures.
Rapid recovery of body mass after surgical removal of adipose tissue in ground squirrels.
Authors: Dark J , Forger N G , Zucker I
Published: May 2006

A substantial proportion of total adipose tissue mass was surgically removed from female ground squirrels during weight gain or weight loss phases of the circannual body weight cycle. Within 2 months of fat removal, squirrels had restored body mass to levels appropriate to the stage of the annual body weight cycle. These findings suggest that ground squirrels may use feedback from body lipids in controlling body weight.
Production of platelet-derived growth factor-like molecules and reduced expression of platelet-derived growth factor receptors accompany transformation by a wide spectrum of agents.
Authors: Bowen-Pope D F , Vogel A , Ross R
Published: May 2006

A series of nontransformed human and murine cells and derivative cell lines transformed by methylcholanthrene; by simian virus 40, Kirsten and Moloney murine sarcoma viruses, simian sarcoma virus, and adenovirus; and by a "spontaneous" event in culture were examined for the expression of receptors for the platelet-derived growth factor (PDGF) and for production of substances able to compete with 125I-labeled PDGF for binding to the cell-surface PDGF receptor. In each case, transformation resulted in a 50-100% decrease in available PDGF receptors. All transformed cells except the methylcholanthrene-transformed mouse cells produce a PDGF competitor into the conditioned medium. Levels of PDGF competitor in conditioned medium at the end of a 48-hr collection were as high as 2 ng/ml--high enough to be measured by radioreceptor assay diluted 1:30 and to maximally stimulate [3H]thymidine incorporation by human fibroblasts. The PDGF competitor activity detected in a radioreceptor assay does not reflect irreversible (e.g., proteolytic) damage to the receptor of test cells since its effects are reversed by acetic acid dissociation. Antiserum against human PDGF neutralizes 20-80% of the PDGF competitor found in conditioned medium from different transformed human cells and 100% of the activity from normal human endothelial cells. The possibility that induction of expression of the cellular PDGF gene may be involved in the mechanism of transformation of PDGF-responsive mesenchymal cells is discussed.
Novel cluster of alpha-interferon gene sequences in a placental cosmid DNA library.
Authors: Lund B , Edlund T , Lindenmaier W , Ny T , Collins J , Lundgren E , von Gabain A
Published: May 2006

A human cosmid library was constructed and probed with a human alpha interferon (IFN-alpha) cDNA clone. One clone giving a strong hybridizing signal was isolated and characterized. The cosmid DNA insert represents a section of the human genome containing three regions of IFN-alpha-like sequences. The DNA was characterized with restriction endonuclease mapping, thereby allowing comparison to similar linkage groups reported recently and determination of homologous regions on the known physical map. The three IFN-alpha-like sequences were analyzed by a partial sequence analysis. Mapping and sequence data establish this section as a not-yet-described cluster of IFN-alpha sequences in the human genome; however, a part of the section matches to some degree to a previously described genomic region. The region described here could represent genetic polymorphism or a duplicated segment.
Hydrolysis of benzo[a]pyrene diol epoxide and its covalent binding to DNA proceed through similar rate-determining steps.
Authors: Meehan T , Bond D M
Published: May 2006

The mutagenic and carcinogenic metabolite of benzo[a]pyrene, (7R,8S)-dihydroxy-(9R,10R)-epoxy-7,8, 9,10-tetrahydrobenzo[a]pyrene, undergoes two major reactions in the presence of DNA: (i) hydrolysis and (ii) covalent binding. We report that hydrolysis and covalent binding are specific and general acid-catalyzed reactions with the same or similar rate-determining steps. To account for the similarity of rate-determining steps in covalent binding and hydrolysis we propose and test two models. In each model, the rate-determining step results in formation of a carbonium ion, which serves as a precursor for both tetrol and adduct. In model A the carbonium ion is partitioned between two domains (1 and 2), while in model B there is only one domain. Measurements of pseudo-first-order rate constants, product ratios, and rate ratios support model A, while kinetic results are inconsistent with model B. Domain 1 most likely represents activated benzo[a]pyrenes that are intercalated into DNA, while domain 2 hydrocarbons are physically bound to the outside of the DNA helix.
Organ-specific nuclear RNAs in tobacco.
Authors: Kamalay J C , Goldberg R B
Published: May 2006

We investigated the developmental regulation of nuclear RNA sequences in tobacco vegetative (leaf, root, stem) and floral (petal, ovary, anther) organ systems using RNA-excess X single-copy DNA hybridization reactions. We found that 18% of the single-copy DNA, equivalent to 1.1 X 10(5) kilobases (kb) of diverse transcripts, is represented in the nuclear RNA of each organ. Each nuclear RNA population has both shared and organ-specific sequences. Depending upon the nuclear RNA, 10-40% of the complexity, or 1.1-4.4 X 10(4) kb of diverse sequence, is organ-specific. Collectively, at least 45% of the single-copy DNA, or 3 X 10(5) kb, is represented in the nuclear RNA of the entire plant. Hybridization experiments with polysomal RNA showed that organ-specific mRNAs are present in both the unique and shared nuclear RNA subsets. Together, our results show that tobacco nuclear RNA sequences are under striking developmental control and that both transcriptional and post-transcriptional processes play a role in regulating plant gene expression.
Induction of immunity to a human tumor marker by in vivo administration of anti-idiotypic antibodies in mice.
Authors: Nepom G T , Nelson K A , Holbeck S L , Hellström I , Hellström K E
Published: May 2006

Anti-idiotypic antibodies are described that were raised against murine monoclonal antibody 8.2, an antibody specific for a human melanoma-associated cell surface marker called p97. The 8.2 idiotopes recognized by this anti-idiotypic antiserum are binding site-associated and are shared by other monoclonal anti-p97 antibodies with the same specificity as antibody 8.2. Mice immunized with the anti-idiotype demonstrate delayed-type hypersensitivity reactions when challenged with melanoma (p97-positive) tumor cells.
Inhibition of tissue damage by the arachidonate lipoxygenase inhibitor BW755C.
Authors: Higgs G A , Mugridge K G , Moncada S , Vane J R
Published: May 2006

The effects of three anti-inflammatory drugs, which interfere with arachidonic acid transformation by three different enzymes, have been compared by using a simple model of tissue damage and foreign body rejection. In groups of control rats, subcutaneously implanted polyester sponges were rejected after a mean of 12 days. Indomethacin, which selectively inhibits prostaglandin synthesis, did not significantly change time to rejection but BW755C (3-amino-1-[m-(trifluoromethyl) phenyl]-2-pyrazoline), which is a dual inhibitor of prostaglandin and leukotriene synthesis, prolonged time to rejection to a mean of 22 days. The anti-inflammatory steroid dexamethasone, which reduces arachidonic acid metabolism by stimulating the formation of a phospholipase inhibitor, prolonged time to sponge rejection as BW755C did. Treatment with BW755C or dexamethasone was also accompanied by a reduction in total leukocyte numbers in inflammatory exudates collected at 1-14 days, whereas indomethacin increased leukocyte migration on days 1 and 2 and had no effect at later times. These results suggest that the inhibition of the leukotriene-forming lipoxygenase suppresses leukocyte activation and that this leads to a reduced rate of tissue damage in experimental inflammation.
Halothane shortens acetylcholine receptor channel kinetics without affecting conductance.
Authors: Lechleiter J , Gruener R
Published: May 2006

The extracellular patch-clamp technique was used to examine how halothane, a general anesthetic, affects the properties of single nicotinic acetylcholine receptor channels of embryonic Xenopus skeletal muscle cells grown in culture. Under control conditions, single-channel events showed a bimodal distribution on the basis of current amplitudes. This distribution was maintained during exposure to halothane and its washout. In addition, the mean current value of the low-and high-amplitude channels was unaffected by the presence of the anesthetic at clinically relevant concentrations. In contrast, halothane shortened the burst durations of both channel types in a concentration-dependent manner. This shortening of burst durations may be an expression of the more rapid relaxation of the channel protein to the nonconducting state, possibly due to the disordering effect of the anesthetic on membrane lipids in which the receptor protein is embedded. This functional change, in the behavior of the synaptic receptor, provides further direct information on the mode of action of general anesthetics.
The presence of a copper/zinc superoxide dismutase in the bacterium Photobacterium leiognathi: a likely case of gene transfer from eukaryotes to prokaryotes.
Authors: Bannister J V , Parker M W
Published: May 2006

The free-living bacterium Photobacterium leiognathi is also known to be a symbiont of ponyfish. The presence of a copper/zinc superoxide dismutase in P. leiognathi has been considered to be a case of gene transfer from eukaryotes to prokaryotes because this form of superoxide dismutase is normally present only in higher eukaryotic species. However, the amino acid sequence of the enzyme from the bacterium exhibited low identities (25-30%) with the same enzyme from eukaryotes. When amino acid mutations are taken into consideration, the weighted sequence similarity increases significantly; furthermore, the bacterial enzyme has the same active site residues and similar predicted secondary structure as the eukaryotic enzymes. The possibility of convergence is ruled out and the case of divergence is considered unlikely because of the observed phylogenetic distribution of the enzyme. This indicates that the presence of the copper/zinc superoxide dismutase in P. leiognathi can indeed be considered a case of gene transfer from eukaryotic species to prokaryotic species.
Transfer of genes into hematopoietic cells using recombinant DNA viruses.
Authors: Karlsson S , Humphries R K , Gluzman Y , Nienhuis A W
Published: May 2006

The ability of recombinant DNA viruses to transfer genes into hematopoietic cells has been explored. A recombinant simian virus 40 (SV40) in which the early region had been replaced with the chloramphenicol acetyltransferase (CAT) gene driven by the promoter from Rous sarcoma virus (RSV), was constructed. This virus transferred the CAT gene more efficiently into mouse and human bone marrow cells and into the K562, MEL, and WEHI hematopoietic tissue culture cell lines, than the classical calcium phosphate DNA transfer procedure, as shown by assay for CAT activity 48 hr after infection. Recombinant SV40 virions were also shown to be capable of stably transforming Chinese hamster ovary cells by use of an early region recombinant containing the methotrexate-resistant dihydrofolate reductase (DHFR) gene driven by the RSV promoter. The entire DHFR transcriptional unit could be detected in the genome of transformed cells that were also shown to be resistant to methotrexate. A recombinant adenovirus stock containing the neomycin-resistance gene driven by the SV40 early promoter was used to infect the K562 and MEL hematopoietic cell lines to resistance to the antibiotic G418. Transformation frequency was 10- to 100-fold higher than that obtained with calcium phosphate-precipitated DNA. Most or all of the recombinant adenovirus genome was integrated as 1-3 copies in the transformed cells. These studies show the feasibility of using DNA viruses for introduction of new genetic material into hematopoietic cells.
Evidence that a second tumor antigen coded by adenovirus early gene region E1a is required for efficient cell transformation.
Authors: Hurwitz D R , Chinnadurai G
Published: May 2006

The expression of the adenovirus (Ad) early coding region 1a (E1a) is required for virus-induced cell transformation and for the activation of other viral early genes and some cellular genes. Two overlapping early mRNAs of 13S and 12S that are transcribed from this region code for a 289-amino acid protein and a 243-amino acid protein, respectively. Earlier studies have shown that the 289-amino acid protein is essential for cell transformation. We have constructed an Ad type 2 (Ad2) deletion mutant (dl231) in which the intervening sequence for the 13S mRNA is precisely removed. Mutant dl231 is completely viable in human KB cells and produces normal amounts of 13S mRNA but much reduced amounts of a defective 12S mRNA. Mutant dl231 induces focal transformation of established rat embryo fibroblasts at a frequency one-fifth to one-half that of wild-type virus. However, the transformed cells are defective in their ability to form anchorage-independent colonies on semisolid medium. Therefore, our results demonstrate that the 243-amino acid protein is required for full transformation of rat embryo cells.
Inversely repeating integrated hepatitis B virus DNA and cellular flanking sequences in the human hepatoma-derived cell line huSP.
Authors: Mizusawa H , Taira M , Yaginuma K , Kobayashi M , Yoshida E , Koike K
Published: May 2006

Among recombinant phages carrying integrated hepatitis B virus (HBV) DNA sequences cloned from the human hepatoma-derived cell line huSP, one clone, lambda hu-489, revealed some unusual features. The 2.25-kilobase Eco D fragment from the insert of this clone hybridized to the HBV DNA probe only and its nucleotide sequence was determined. The viral sequence, as well as a cellular flanking sequence, showed extensive rearrangement accompanied by inverted repetition. The Eco D fragment contained HBV DNA from the 5'-end region of gene S to the middle of gene X, followed by a long cellular flanking sequence. Moreover, a part of gene X was found inversely repeated at the head of the same gene S in a head-to-head configuration truncated by the same cellular sequence. Therefore, the same junction sequence of viral DNA and the cellular sequence was found at two different sites in the Eco D fragment in opposite polarities.
Genetic rearrangement of DNA induces knots with a unique topology: implications for the mechanism of synapsis and crossing-over.
Authors: Griffith J D , Nash H A
Published: May 2006

We have determined the topological sign of the knots produced by a cycle of phage lambda integrative recombination. To insure that these knots reflect intrinsic features of the reaction mechanism, the substrate was constructed so that random interwrapping of segments of DNA played a minimal role in the topological outcome. The knotted DNA was coated with the bacteriophage T4 uvsX gene product and examined in the electron microscope to determine the nature of each crossing point or node. All of the knots were identical; they were trefoils with three nodes of positive sign. We interpret this result to mean that one recombination site, which previous work had indicated is organized into a nucleosome-like structure, is wrapped with a handedness identical to that found in nucleosomes. Therefore, this wrapping may explain the dependence of recombination on supercoiling of the substrate DNA. Moreover, we show that the topological result sharply limits acceptable mechanisms for the details of strand exchange.
A neutron diffraction study on the location of the polyene chain of retinal in bacteriorhodopsin.
Authors: Seiff F , Wallat I , Ermann P , Heyn M P
Published: May 2006

We report on the location of the chain part of the retinylidene chromophore in the projected density of bacteriorhodopsin as determined by neutron diffraction from the two-dimensional purple membrane lattice. For this purpose, partially deuterated retinal was synthesized containing 10 deuterons at positions C-8, C-10, C-12, C-14, C-19(3), and C-20(3) of the polyene chain. Two sets of dark-adapted samples were prepared in entirely different ways: (i) Deuterated retinal was incorporated biosynthetically during growth of the bacteria by using the mutant JW5, which is deficient in the synthesis of retinal. (ii) The chromophore was converted to retinal oxime, the resulting colorless apomembrane was regenerated with deuterated retinal, and the residual retinal oxime was removed by washing with bovine serum albumin. Characterization of these samples by x-ray diffraction, absorption, and flash spectroscopy showed that they were identical to native purple membrane samples as judged by these criteria. Fourier difference maps were calculated from the differences in inplane diffraction from the deuterated membranes and from protonated samples that were prepared in exactly the same way. At 8.7 A resolution, both maps show a single major peak at the same position with the center of mass of the labeled part of the chain (C-11) between helices 6 and 3 but closer to helix 6. It appears likely that the COOH-terminal helix G, to which retinal is attached at lysine-216, is either helix 2 or 6.