bluuebunny/crossref_metadata_2025 huggingface dataset:
Malignant transformation induced by incorporated radionuclides in BALB/3T3 mouse embryo fibroblasts.
Authors: LeMotte P K , Adelstein S J , Little J B
Published: May 2006

The induction of lethality and malignant transformation by 5-[125I]iododeoxyuridine and [3H]thymidine incorporated into cellular DNA and by x-irradiation was studied in vitro in BALB/3T3 cells. Under these conditions, 125I radiation is highly localized to small regions of the DNA at the site of each decay and produces DNA double-strand breaks with high efficiency. Incorporated 125I was found to be 12-16 times as lethal per decay as incorporated 3H. For the induction of malignant transformation, however, 125I was approximately 25 times as effective per decay as 3H. When the frequencies of transformation induced at various levels of survival by 125I, 3H, and x-rays were compared, lethality was found to correlate closely with transformation at doses that yielded significant cell killing. An exception occurred at low doses, where 125I appeared much more efficient than x-irradiation in inducing transformation; transformation frequencies equal to those induced by 3-5 Gy of x-rays resulted from 125I exposures that yielded little or no cell killing.
Desensitization of steroidogenesis in cultured Leydig tumor cells: Role of cholesterol
Authors: Freeman Dale A. , Ascoli Mario
Published: May 2006

We have reported previously that the human choriogonadotropin (hCG) receptors of cultured Leydig tumor cells can be down-regulated with hCG or mouse epidermal growth factor (mEGF) and that such down-regulation results in a loss of the steroidogenic response of the cells to hCG. Exposure of the cells to hCG, but not to mEGF, also resulted in a decrease in steroidogenic responses to cholera toxin and cAMP. The results presented herein show that the hCG-induced loss of steroidogenic response to cAMP is due to the depletion of intracellular cholesterol and that this depletion can be prevented by the addition of low density lipoprotein. Our results also show that after exposure of the cells to mEGF or hCG their steroidogenic response to hCG is limited by the number of hCG receptors, regardless of the presence of low density lipoprotein.
Human c-myc onc gene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells.
Authors: Dalla-Favera R , Bregni M , Erikson J , Patterson D , Gallo R C , Croce C M
Published: May 2006

Human sequences related to the transforming gene (v-myc) of avian myelocytomatosis virus (MC29) are represented by at least one gene and several related sequences that may represent pseudogenes. By using a DNA probe that is specific for the complete gene (c-myc), different somatic cell hybrids possessing varying numbers of human chromosomes were analyzed by the Southern blotting technique. The results indicate that the human c-myc gene is located on chromosome 8. The analysis of hybrids between rodent cells and human Burkitt lymphoma cells, which carry a reciprocal translocation between chromosomes 8 and 14, allowed the mapping of the human c-myc gene on region (q24 leads to qter) of chromosome 8. This chromosomal region is translocated to either human chromosome 2, 14, or 22 in Burkitt lymphoma cells.
Cloning and nucleotide sequence of mouse immunoglobulin epsilon chain cDNA.
Authors: Liu F T , Albrandt K , Sutcliffe J G , Katz D H
Published: May 2006

cDNA corresponding to mouse IgE heavy (epsilon) chain mRNA was cloned from mouse IgE-secreting hybridoma cells. A clone containing the epsilon cDNA insert was identified by hybridization to epsilon mRNA and subsequent translation in vitro to unprocessed epsilon chain reactive with anti-mouse IgE antibodies. This clone was used to select 20 other epsilon cDNA clones by colony hybridization. The clone containing the longest insert was selected and the epsilon cDNA insert was subjected to sequence analysis. The determined sequence is 1,279 nucleotides long and contains the coding regions for part of the constant region (C epsilon) I and all of the C epsilon 2, C epsilon 3, and C epsilon 4 domains and also the entire 3' untranslated region of epsilon mRNA. When the amino acid sequence determined from the nucleotide sequence is compared to that of human epsilon chain, significant homologies between corresponding domains of the two epsilon chains are found, including conservations in cysteine and tryptophan residues and carbohydrate attachment sites.
Growth hormone releasing factor, somatocrinin, releases pituitary growth hormone in vitro.
Authors: Brazeau P , Ling N , Böhlen P , Esch F , Ying S Y , Guillemin R
Published: May 2006

Purified (rat) hypothalamic growth hormone releasing factor (GRF), native human GRF isolated from an islet cell tumor of the pancreas that had caused acromegaly, and the synthetic replicates of the human material are potent secretagogues of immunoreactive growth hormone (GH) by primary cultures of rat pituitary cells. Native or synthetic peptides give identical dose-response curves, with identical slopes and identical maximal effects. The median effective dose of the tumor-derived GRF is 15 x 10(-12) M. The effect of hypothalamic GRF or of a synthetic replicate of tumor-derived GRF is immediate, being demonstrable in less than or equal to 30 sec after contact in a pituitary cell perifusion system. The effect of hypothalamic GRF or of tumor-derived GRF is highly specific for stimulating release of immunoreactive growth hormone; there is no demonstrable concomitant effect on the secretion of other pituitary hormones. Somatostatin-28 and somatostatin-14 inhibit the release of growth hormone produced by hypothalamic GRF or tumor-derived GRF in typical noncompetitive antagonism. On the basis of the results reported here, hypothalamic GRF and tumor-derived GRF are qualitatively indistinguishable in their ability to stimulate the secretion of immunoreactive growth hormone in vitro. The name "somatocrinin" is proposed to replace the acronym GRF.
Inequalities for ratios of functionals of harmonic functions
Authors: Fefferman R. , Gundy R. , Silverstein M. , Stein E. M.
Published: May 2006

We show that the ratios (
A
(
u
)/
N
(
u
))
k
A
(
u
) and (
N
(
u
)/
A
(
u
))
k
N
(
u
) have

L
p

norms equivalent with the

H
p

norm of
u
. Here
A
(
u
) is the Lusin area integral and
N
(
u
) is the nontangential maximal function.
Cytochrome c gene-related sequences in mammalian genomes.
Authors: Scarpulla R C , Agne K M , Wu R
Published: May 2006

We use a rat cytochrome c gene that we previously isolated and determined the sequence of to estimate the number of related sequences present in the rat genome. Approximately 25 different EcoRI restriction endonuclease fragments from total rat DNA hybridize to the gene of known structure. Four of these correspond to homologous sequences present in four different lambda Charon 4A-rat cytochrome c recombinants previously isolated. Intact or nearly intact genes appear to reside on almost all of the genomic fragments, because they hybridize strongly to gene subfragments representing both 5' and 3' portions of the coding sequence as well as to 3' noncoding DNA that is found specifically associated with the coding region. A subgroup of about six of the fragments also shares homology within the 73 nucleotides immediately preceding the AUG codon. An intron-specific probe reveals only the EcoRI fragment from which it was derived and one other genomic fragment. On the basis of the temperature of complete dissociation of the coding region probe in 0.75 M NaCl/0.075 M Na3 citrate/50% (vol/vol) formamide, the 25 fragments are separable into three stringency classes of 40-50 degrees C, 50-55 degrees C, and 55-60 degrees C. The latter, high-stringency group of about seven fragments includes those cloned in the recombinant phage isolates, whose regions homologous to cytochrome c are shown to differ from the purified gene of known sequence by an amount equivalent to about 2% mismatched bases. Families of cytochrome c gene-related sequences are also found in the genomes of several other mammals, including humans.
Microtubules and microfilaments in fixed and permeabilized cells are selectively decorated by nerve growth factor.
Authors: Nasi S , Cirillo D , Naldini L , Marchisio P C , Calissano P
Published: May 2006

A specific antibody against nerve growth factor (NGF) and indirect immunofluorescence microscopy have been used to follow the in vitro binding of NGF to cells made permeable to large molecules. All cells tested, both target (sensory neurons and PCI2 cells) and nontarget (3T3, BKH 2I, C6 glioma cells), revealed a decoration of cytoskeletal structures which on the basis of their form, reactivity with antibodies, and sensitivity to specific drugs may be identified as microtubules (MTs) and microfilaments (MFs). The decoration of either structure depends on the fixation and permeabilization conditions: MFs, in the form of stress fibers, are stained by NGF when the plasma membrane is permeabilized with methanol/acetone; MTs become intensely stained when the plasma membrane is solubilized with a nonionic detergent in the presence of a MT-stabilizing medium. The two procedures do not affect the staining of these structures with specific antibodies. Binding of 125I-labeled NGF to PCI2 cells was not competitively inhibited by a 100-fold excess of several positively charged proteins but it was markedly decreased in the presence of DNase I. 125I-Labeled NGF interacted with MTs and F-actin (fixed with paraformaldehyde) in a range of concentrations similar to that used for their cellular localization with NGF-anti-NGF. Our studies show that the specificity and affinity of NGF binding to MTs and MFs is in the range of that of antibodies against tubulin and actin. The possible relevance of these findings to the mechanism of action of NGF in target cells is discussed.
Circadian rhythms in Neurospora crassa: a mutation affecting temperature compensation.
Authors: Mattern D L , Forman L R , Brody S
Published: May 2006

The circadian rhythm of conidiation (spore formation) in Neurospora crassa is known to be temperature compensated, that is, the period is only slightly affected by the incubation temperature. Thus, the Q10 (the relative rate enhancement corresponding to a 10 degrees C rise in temperature) of the rhythm of the bd csp strain from 14 to 30 degrees C was 1.1, whereas the Q10 of the uncompensated growth rate in the same interval was 2.4. A mutation at the cel locus resulted in loss of the temperature-compensation property in cultures grown below 22 degrees C. The Q10 of the rhythm below 22 degrees C was 2.2, and periods of about 40 hr were observed. In contrast, the Q10 of the rhythm above 22 degrees C was 1.1, with circadian periods of 18-21 hr. Thus, cel displayed a threshold temperature or "break point" for the temperature compensation of its rhythm. Supplementation of cel strains, which require fatty acids, with unsaturated or short-chain fatty acids raised the threshold temperature to 26 degrees C, whereas supplementation with long-chain saturated fatty acids lowered it to 18 degrees C. These data suggest a role for fatty acids, as liquid components or as cellular metabolites, in the mechanism of temperature compensation.
Specific cellular stimulation in the primary immune response: experimental test of a quantized model.
Authors: Dintzis R Z , Vogelstein B , Dintzis H M
Published: May 2006

Dose-response and dose-suppression curves have been measured for the primary immune response in mice, in vivo and in vitro, by using size-fractionated linear polymers of acrylamide substituted with hapten. The results are in general agreement with a simple theory based on the premise that the specific primary immunological response is quantized at some fundamental and limiting step, requiring a minimum number of linked antigen receptors for response.
Osmotic regulation and the biosynthesis of membrane-derived oligosaccharides in Escherichia coli.
Authors: Kennedy E P
Published: May 2006

The membrane-derived oligosaccharides (MDO) of Escherichia coli are periplasmic constituents containing 8-10 glucose units in a highly branched structure, linked by beta 1-2 and beta 1-6 bonds [Schneider, J. E. Reinhold, V., Rumley, M. K. & Kennedy, E. P. (1979) J. Biol. Chem. 254, 10135-10138]. The MDO are multiply substituted with sn-1-phosphoglycerol residues (derived from membrane phosphatidylglycerol) and with O-succinyl ester residues and, thus, are high anionic. Experiments in this paper offer evidence that the biosynthesis of MDO is an important aspects of osmoregulation in E. coli. Cells grown in medium of low osmolarity (ca. 50 mosM) synthesize 16 times more MDO than those grown in the same medium with 0.4 M NaCl. In cells grown in medium of low osmolarity, it appears that MDO is the principal source of fixed anion in the periplasmic space and, thus, acts to maintain the high osmotic pressure and Donnan membrane potential of the periplasmic compartment Regulation of MDO synthesis in response to changes in osmolarity of the medium appears to occur at the genetic level because the synthesis of new protein is needed to permit the production of MDO at high rates after shift of cells to medium of low osmolarity.
Purification and properties of the uvrA protein from Escherichia coli.
Authors: Seeberg E , Steinum A L
Published: May 2006

The uvrA+ gene product from Escherichia coli was purified to apparent homogeneity; the assay measured its ability to restore repair endonuclease activity in extracts from uvrA mutated cells. The uvrA protein is a 115,000 molecular weight DNA-binding protein having higher affinity for single-stranded than double-stranded DNA. It does not introduce single-strand breaks or alkali-labile bonds in native or UV-irradiated DNA, but it catalyzes hydrolysis of ATP to ADP and Pi. The ATPase activity is not DNA dependent and has a Km of 0.23 mM, which corresponds to the Km for the ATP requirement of the UV-endonuclease reaction catalyzed by the combined uvrA+, uvrB+, and uvrC+ gene products. ADP and adenosine 5'-[gamma-thio]triphosphate both inhibit the uvrA ATPase as well as the uvrABC endonuclease and also prevent specific binding of the uvrA proteins to UV-irradiated DNA. These results indicate that both the DNA-binding property and the ATPase activity of the uvrA protein are essential for uvrABC endonuclease activity and that the ATP requirement of the endonuclease reaction is determined by uvrA ATPase.
Hydrolysis of human platelet membrane glycoproteins with a
Serratia marcescens
metalloprotease: Effect on response to thrombin and von Willebrand factor
Authors: Cooper Herbert A. , Bennett William P. , White Gilbert C. , Wagner Robert H.
Published: May 2006

Metalloproteases from at least two Gram-negative organisms selectively hydrolyze the surface of human fixed washed platelets. The protease-treated platelets lose their ability to aggregate with bovine von Willebrand factor and human von Willebrand factor plus ristocetin. The present study reports the membrane glycoprotein alterations and loss of platelet function that occur after incubation of fresh washed human platelets with a purified protease produced by
Serratia marcescens
. The studies were undertaken in order to examine, using the
Serratia
protease as an enzymatic probe, the relationship between externally oriented glycoproteins and two known aggregating agents, bovine von Willebrand factor and thrombin. Platelet membrane glycoproteins were analyzed after discontinuous NaDodSO
4
/polyacrylamide gel electrophoresis using both staining with periodic acid-Schiff reagent and autoradiography of
3
H-labeled platelets. Both methods of detection demonstrated that at
Serratia
protease concentrations above 0.6 μg/ml there was hydrolysis within 3 min of a membrane glycoprotein (
M
r
185,000) corresponding to GPIb in control platelets. The loss of GPIb was accompanied by the appearance in the platelet supernatant of a glycopeptide (
M
r
156,000). Under reducing conditions, the hydrolyzed membrane glycopeptide in the supernatant had a consistently faster migration (
M
r
149,000). Platelets treated with
Serratia
protease at concentrations sufficient to give maximal cleavage of GPIb were unresponsive to bovine von Willebrand factor and did not aggregate or release [
14
C]serotonin at doses of von Willebrand factor up to 0.33 unit/ml. On the other hand, the response to bovine α-thrombin was only minimally impaired by treatment with
Serratia
protease. These results implicate GPIb in the response by platelets to bovine von Willebrand factor but suggest that surface components other than GPIb play a major role in the response by platelets to thrombin.
Use of different tRNASer isoacceptor species in vitro to discriminate between the expression of plasmid genes.
Authors: Cenatiempo Y , Robakis N , Meza-Basso L , Brot N , Weissbach H , Reid B R
Published: May 2006

A simplified translation system coupled to DNA transcription that involves assaying the synthesis of the first dipeptide of a gene product has been described recently [Robakis, N., Meza-Basso, L., Brot, N. & Weissbach, H. (1981) Proc. Natl. Acad. Sci. USA 78, 4261--4264]. Using this dipeptide system, we have investigated the expression of genes carried on plasmids coding for beta-lactamase, ribosomal protein L12, and the chloroplast large subunit (LS) of ribulosebisphosphate carboxylase (RbuBPCase). Although all three nascent gene products begin with the sequence fMet-Ser, the formation of fMet-Ser can be used to distinguish between the synthesis of beta-lactamase and either L12 or the LS of RbuBPCase by using different serine isoacceptor tRNA species. In beta-lactamase, the serine codon is AGU, which utilizes the serine isoacceptor species tRNASer3; in L12 and the LS of RbuBPCase, the serine codewords are UCU and UCA, respectively, both of which are recognized by the serine isoacceptor species tRNASer1. By using either pure tRNASer1 or pure tRNASer3, the expression of each gene can be quantitated. In this system, guanosine-5'-diphosphate-3'-diphosphate inhibits the expression of the beta-lactamase and L12 genes but stimulates the synthesis of the LS. In addition, the ratio of fMet-Ser/fMet-Ala (L12/L10) synthesized was about 1 as compared with the ratio of 4 that has been obtained previously in vivo or in vitro protein-synthesizing systems in which the entire gene product was measured.
H-2Kk and vesicular stomatitis virus G proteins are not extensively associated in reconstituted membranes recognized by T cells.
Authors: Cartwright G S , Smith L M , Heinzelmann E W , Ruebush M J , Parce J W , McConnell H M
Published: May 2006

It is shown that liposomes containing (i) a fluorescein-labeled murine histocompatibility antigen (FITC-H-2Kk) and the G protein of vesicular stomatitis virus or (ii) H-2Kk and fluorescein-labeled viral protein (FITC-G) can elicit H-2-restricted syngeneic antiviral cytotoxic T cells as assayed by 51Cr release from appropriate virus-infected target cells. Fluorescence recovery after photobleaching was used to measure the diffusion coefficients of these reconstituted proteins in four different samples: (i) FITC-H-2Kk; (ii) FITC-H-2Kk and G; (iii) FITC-G; and (iv) FITC-G and H-2Kk. The same rate of lateral diffusion (D = 1 x 10(-8) cm2/sec at 37 degrees C in 25% cholesterol/75% dimyristoylphosphatidylcholine) was obtained in every case. Both proteins, fluorescent as well as nonfluorescent, could be patched by using specific antibodies. When G was patched with antibody, FITC-H-2Kk did not copatch. When H-2Kk was patched with antibody FITC-G did not copatch. These diffusion and patching measurements rule out the possibility that these proteins have either extensive oligomeric associations or strong specific pairwise associations.
Molecular genetics of vaccinia virus: demonstration of marker rescue.
Authors: Nakano E , Panicali D , Paoletti E
Published: May 2006

Two genomic variants of vaccinia virus isolated from serially propagated stocks were used to demonstrate marker rescue. The smaller (S variant) virus contains a 6.3 megadalton (MDal) deletion of unique DNA sequences present in the 123-MDal larger (L variant) virus. The deletion was mapped at 6.85 MDal from the left terminus of the genome, just outside of the inverted terminal repetition. Rescue of the unique deleted DNA sequences by infectious S variant virus was obtained in CV-1 cells by using the calcium orthophosphate precipitation technique of intact or restriction endonuclease-treated L-variant DNA. Restriction fragments that overlapped the deletion allowed marker rescue, but restriction of the L-variant DNA within the unique deleted sequences gave negative results. Restriction endonuclease analysis of the DNA obtained from twice-plaque-purified recombinant virus derived from the rescue of overlap donor fragments gave a restriction pattern identical to that of L-variant virus, indicating that the donor DNA was inserted into the rescuing virus by double recombination. No amplification of the unique sequences was observed from intact L-variant DNA in the absence of infectious S-variant virus, suggesting that deproteinized vaccinia DNA is noninfectious and that the donor DNA was neither integrated into the host DNA nor present as an episomal structure. By using 1 microgram of intact L-variant DNA per CV-1 monolayer in a 6-cm Petri dish, approximately 1--5% of the plaques contained the L-variant genotype, and the dose--response curve was essentially linear from 0.1 to 2 microgram of DNA.
Identification of a novel sex steroid binding protein.
Authors: Taylor R N , Smith R G
Published: May 2006

We describe a cytoplasmic steroid binding protein in the chick oviduct which has intriguing characteristics. This protein binds [3H]estradiol with high affinity (Kd = 30 X 10(-9) M) and limited capacity (300 fmol/mg of cytosol protein). It sediments at 8 S in low-salt sucrose density gradients and at 4 S in high-salt gradients. Unlike the estrogen receptor, however, this protein also binds progesterone, R5020, testosterone, and 5 alpha-dihydrotestosterone with similar affinities and in a competitive manner. Moreover, it is not translocated to the nucleus by the in vivo administration of these sex steroids. The protein is only present in estrogen-responsive tissue, and like the sex steroid receptors, its synthesis appears to be regulated by estrogen.
Photoaffinity-labeling and fluorescence-distribution studies of gonadotropin-releasing hormone receptors in ovarian granulosa cells.
Authors: Hazum E , Nimrod A
Published: May 2006

Photoaffinity labeling of rat ovarian granulosa cells and membrane preparations with a bioactive photoaffinity derivative of gonadotropin-releasing hormone resulted in identification of two specific components with apparent molecular weights of 60,000 and 54,000. Fluorescent visualization of gonadotropin-releasing hormone receptors in these cells, by using a bioactive rhodamine derivative of the hormone, indicated that the fluorescently labeled receptors were initially distributed uniformly on the cell surface and then formed patches that subsequently internalized (at 37 degrees C) into endocytic vesicles. These processes were dependent on specific binding sites for the rhodamine-labeled peptide on the granulosa cells. These studies may provide an experimental basis for understanding the molecular events involved in the action of the hormone in the ovary.
Purification of two subspecies of human gamma (immune) interferon.
Authors: Yip Y K , Barrowclough B S , Urban C , Vilcek J
Published: May 2006

Interferon (IFN)-gamma was produced in cultures of human leukocytes by combined stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA) and phytohemagglutinin (PHA). IFN-gamma was purified by sequential adsorption and elution from controlled-pore glass and concanavalin A-Sepharose and by subsequent adsorptive removal of contaminating proteins on DEAE-Sephacel at pH 8.0. Treatment of such partially purified IFN-gamma preparations with the anionic detergent NaDodSO4 (0.1% at 20-25 degrees C) decreased biological activity to approximately 5-20%. When analyzed by NaDodSO4/polyacrylamide gel electrophoresis the bulk of IFN activity not destroyed by NaDodSO4 treatment was recovered from two peaks with apparent molecular weights of 20,000 and 25,000. The two activity peaks showed close correspondence with Coomassie blue-stained bands regularly demonstrable in purified supernatants from induced cultures but absent from culture supernatants from uninduced cells. Available evidence suggests that the two bands, isolated in pure form, represent subspecies of IFN-gamma. Native IFN-gamma was found to have a lower affinity for alkyl agarose columns than human IFN-alpha or IFN-beta did, suggesting that IFN-gamma is a relatively hydrophilic protein. Sulfhydryl-specific binding of native IFN-gamma to an Affi-Gel 501 column suggested that this IFN contains free sulfhydryl.
Visualization of microtubules in interphase and mitotic plant cells of Haemanthus endosperm with the immuno-gold staining method.
Authors: De Mey J , Lambert A M , Bajer A S , Moeremans M , De Brabander M
Published: May 2006

A procedure is presented for the immunocytochemical visualization of microtubules in interphase and mitotic cells of Haemanthus endosperm. It includes preservation of microtubules (MTs) with glutaraldehyde and uses colloidal gold, coated with secondary antibodies, in a novel indirect-light microscopic technique: the immuno-gold staining method. This immunocytochemical stain allows us to follow the changes in distribution of MTs during mitosis with greater precision and specificity than allowed by other light microscopic techniques. Many aspects of MT arrangements, as reported from ultrastructural studies, are corroborated and extended. This demonstrates the reliability of the technique. In addition, a number of significant observations were made. These concern (i) the presence of a network of MTs in interphase cells, (ii) the transformation of this network into a spindle-like cage of MTs (the clear zone) surrounding the nucleus during prophase, (iii) the drastic rearrangement of MT distribution during prometaphase, (iv) new evidence for the formation of aster-like arrays of polar MTs during anaphase, and (v) the development of the phragmoplast.
Linkage and sequence homology of two human immunoglobulin gamma heavy chain constant region genes.
Authors: Ellison J , Hood L
Published: May 2006

We report the nucleotide sequence of a gene encoding a human immunoglobulin C gamma 2 region. Comparison with the previously determined C gamma 4 sequence reveals that these two genes share extensive (approximately 95%) homology in the three CH domain exons and adjacent noncoding regions. In contrast, hinge exons have diverged to a much greater degree, implying that natural selection has favored the generation of diversity in these coding regions. We have used the noncoding nucleotide differences to estimate that approximately 6-7 million years have elapsed since the occurrence of the gene duplication or correction event which generated the two identical ancestral genes. In addition we show that the two C gamma genes are arranged in human chromosomal DNA in the configuration 5'-C gamma 2-17 kilobase pairs -C gamma 4-3'.
Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin.
Authors: Eisinger M , Marko O
Published: May 2006

Cultures consisting almost entirely of human melanocytes were obtained from epidermal single-cell suspensions by using phorbol 12-myristate 13-acetate (10 ng/ml) in the culture medium. At this concentration, phorbol ester is toxic to human keratinocytes but not to melanocytes. When the seeding density was optimal (0.8-2 x 10(4)/cm2) and the medium contained both phorbol ester and cholera toxin, melanocytes proliferated extensively. Under these conditions, human melanocytes could be passaged serially in vitro and grown in quantity. This cell culture system can thus be used to answer basic questions related to pigment cell biology and may serve as a control for studies of malignant melanocytes.
Glucocorticoid hormone resistance during primate evolution: receptor-mediated mechanisms.
Authors: Chrousos G P , Renquist D , Brandon D , Eil C , Pugeat M , Vigersky R , Cutler G B , Loriaux D L , Lipsett M B
Published: May 2006

The concentrations of total and protein-unbound plasma cortisol of New World monkeys are higher than those of Old World primates and prosimians. The urinary free-cortisol excretion also is increased markedly. However, there is no physiologic evidence of increased cortisol effect. These findings suggest end-organ resistance to glucocorticoids. This was confirmed by showing that the hypothalamic-pituitary adrenal axis is resistant to suppression by dexamethasone. To study this phenomenon, glucocorticoid receptors were examined in circulating mononuclear leukocytes and cultured skin fibroblasts from both New and Old World species. The receptor content is the same in all species, but the New World monkeys have a markedly decreased binding affinity for dexamethasone. Thus, the resistance of these species to the action of cortisol is due to the decreased binding affinity of the glucocorticoid receptor. This presumed mutation must have occurred after the bifurcation of Old and New World primates (approximately 60 x 10(6) yr ago) and before the diversion of the New World primates from each other (approximately 15 x 10(6) yr ago).
Batrachotoxin modifies the gating kinetics of sodium channels in internally perfused neuroblastoma cells.
Authors: Huang L Y , Moran N , Ehrenstein G
Published: May 2006

We have studied the effects of batrachotoxin (BTX) on sodium channels in hybrid mouse neuroblastoma cells NG108-15 by using the suction pipet voltage clamp method. BTX-modified sodium channels activate with first-order kinetics and, over most of the potential range, activate more slowly than normal sodium channels. The peak conductance-voltage curve and the time constant of activation-versus-voltage curve for BTX-modified sodium channels are shifted about 50 mV in the hyperpolarizing direction compared to the corresponding curves for normal sodium channels. There is no change in the slope of the conductance-voltage curve. These results suggest that BTX slows down one of the steps leading to channel opening, which consequently becomes rate-limiting. In addition, BTX eliminates both fast and slow inactivation.
Corticotropin and β-endorphin-like materials are native to unicellular organisms
Authors: LeRoith D. , Liotta A. S. , Roth J. , Shiloach J. , Lewis M. E. , Pert C. B. , Krieger D. T.
Published: May 2006

Multiple molecular forms of immunoreactive corticotropin (ACTH) and β-endorphin were present in extracts of a unicellular eukaryote (
Tetrahymena pyriformis
). One form of immunoreactive ACTH reacted similarly with two different ACTH antisera (one specific for the 11-24 sequence and the other with determinants within sequences 1-14 and 17-39) and migrated with synthetic hACTH-(1-39) in a gel filtration system. This form also exhibited ACTH bioactivity in a dispersed rat adrenal cell bioassay system, with a mean immunoassay/bioassay ratio of 1.5. Gel filtration revealed multiple size classes of immunoreactive β-endorphin; a major peak of radioreceptor activity was detected which exhibited a K
av
similar to that of authentic β-endorphin. A major portion of immunoreactive β-endorphin-sized material exhibited retention times similar to those of synthetic human and camel β-endorphin upon reverse-phase high-pressure liquid chromatography. These distinctive properties and specificities would seem to exclude the presence of limited homologies with sequences present in other proteins. High molecular weight material containing both ACTH and β-endorphin antigenic determinants was also demonstrated, suggesting, but not proving, the presence of a common precursor molecule.
General phase-invariant formulas of higher order: Expected values
Authors: Karle Jerome
Published: May 2006

Formulas for the cosines of the higher order phase invariants that arise in crystal structure analysis are derived as expected values from determinantal joint probability distributions. The values of the cosines of the invariants are expressed in terms of averages over simple functions of known structure factor magnitudes. The formulas are termed “general” to distinguish them from formulas that have more restricted averages and are termed “special.” As with the special formulas, the best opportunity for obtaining useful information from these formulas is provided by embedded seminvariants formed from the invariants by use of relationships among the phases that arise from the space group symmetries.
Stochastic games have a value
Authors: Mertens Jean-Francois , Neyman Abraham
Published: May 2006

Undiscounted nonterminating stochastic games in which the state and action spaces are finite have a value.
Biochemical comparison of major histocompatibility complex molecules from different subspecies of Mus musculus: evidence for trans-specific evolution of alleles.
Authors: Arden B , Klein J
Published: May 2006

H-2 haplotypes were extracted from wild mice of three subspecies, Mus musculus domesticus, M. m. molossinus, and M. m. castaneus, that are known to have been separated from one another for some 1 to 2 million years. Serologically indistinguishable molecules controlled by some of the polymorphic H-2 loci were compared by tryptic peptide mapping, and the maps were found to be identical. In addition, a number of instances of biochemically indistinguishable H-2 molecules were found among wild mice and inbred strains of the M. m. domesticus subspecies. These findings suggest that some of the H-2 alleles have not altered for greater than 1 million years. To reconcile this apparent stability of H-2 genes with their extraordinary polymorphism (some 100 alleles at each of the polymorphic H-2 loci), it is proposed that the H-2 alleles evolve as if they were separate loci.
Transformation with DNA from 5-azacytidine-reactivated X chromosomes.
Authors: Venolia L , Gartler S M , Wassman E R , Yen P , Mohandas T , Shapiro L J
Published: May 2006

It has been shown that 5-azacytidine (5-Aza-Cyd) can reactivate genes on the inactive human X chromosome. It is assumed that the 5-Aza-Cyd acts by causing demethylation of the DNA at specific sites, but this cannot be demonstrated directly without a cloned probe. Instead, we have utilized the technique of DNA-mediated transformation to show that the 5-Aza-Cyd-induced reactivation occurs at the DNA level. DNAs from various mouse-human or hamster-human hybrid cell lines, deficient for mouse or hamster hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8) and varying in whether they contained either an active or inactive human X chromosome, were used in transformation of HPRT- cells. DNA from the active human X chromosome-containing cell lines yielded HPRT+ transformants, whereas DNA from the inactive X chromosome-containing cells lines did not. The inactive X chromosomal DNA was able to transform thymidine kinase-deficient mouse cells, indicating that the DNA solution was normal. These results confirm that inactivation of the X chromosome involves a DNA modification. Furthermore, DNAs from three cell lines with a 5-Aza-Cyd-reactivated X chromosome also transform HPRT- cells, demonstrating that the 5-Aza-Cyd has altered the DNA structure and supporting the idea that methylation plays a role in X chromosome inactivation.
Interactions between molecules (subfactors) released by different T cell sets that yield a complete factor with biological (suppressive) activity.
Authors: Ptak W , Rosenstein R W , Gershon R K
Published: May 2006

T cells that have been immunized to express optimal levels of contact hypersensitivity upon adoptive transfer to normal animals can be inhibited from doing so by incubating them with an antigen-specific T suppressor factor. This factor is composed of at least two subunits which come from cells expressing different Ly phenotypes; an antigen-specific antigen-binding "subfactor" is made by an Ly-1 cell and a non-antigen-binding one is made by an Ly-2 cell. Neither of these cells nor their products express detectable amounts of major histocompatibility gene products. The mode of immunization plays an important role in determining which of these subfactors will be produced. Painting the skin with a reactive hapten immunizes Ly-1 cells that secrete antigen-binding material, whereas intravenous injection of trinitrobenzenesulfonic acid activates Ly-2 cells to produce a second subunit that does not see antigen. There is reason to believe that the molecule that does not bind to antigen does have some antigen specificity. An analysis of the data at hand suggests that the antigen specificity stems from an interaction of the two subunits described with yet another subunit and that biological activity is dependent upon three macromolecules. Thus, the complex level of cellular interactions that regulate immunity may also be reflected in a similar type of complexity in the interaction between their biologically active cell-free products.
The cytochrome P-450-depleted animal: an experimental model for in vivo studies in chemical biology.
Authors: Drummond G S , Kappas A
Published: May 2006

An experimental method is described to deplete markedly in vivo the cytochrome P-450 content of liver for prolonged periods of time. The method uses the synthetic metalloporphyrin cobalt-heme (cobalt protoporphyrin IX), which possesses the dual biological properties of repressing delta-aminolevulinate synthase, the rate-limiting enzyme of heme biosynthesis, and of potently inducing microsomal heme oxygenase, the rate-limiting enzyme of heme catabolism. A single dose of cobalt-heme (125 mumol/kg of body weight) decreased within 48 hr hepatic cytochrome P-450 to approximately 20% of normal, at which level it remained for 10 days; normal levels were not achieved by 36 days. Periodic administration (total, six injections) of a smaller dose of cobalt-heme (50 mumol/kg of body weight) maintained the cytochrome P-450 content at levels approximately 15% of normal for greater than 90 days with concurrent profound impairment of mono-oxygenase reactions catalyzed by this heme protein. The ability of cobalt-heme to produce profound and prolonged depletion of cytochrome P-450 in vivo provides a valuable model for examining the role of cytochrome P-450-dependent metabolism in the biology of endogenous and exogenous chemicals.
Immunocytochemical localization of enkephalin-like immunoreactivity in the retina of the guinea pig.
Authors: Altschuler R A , Mosinger J L , Hoffman D W , Parakkal M H
Published: May 2006

The distribution of enkephalin-like immunoreactivity in the retina of the guinea pig was studied. Indirect immunofluorescence techniques were used on retinae with and without colchicine pretreatment. In retinae not receiving colchicine pretreatment, enkephalin-like immunoreactivity was seen in fibers in the inner plexiform layer, predominantly in laminae 1, 3, and 5. In colchicine-pretreated retinae, enkephalin immunofluorescent cell bodies were seen in the inner margin of the inner nuclear layer in addition to the immunoreactive fibers. These cells showed morphological characteristics of amacrine cells. No enkephalin-like immunofluorescence was seen in the optic nerve, ganglion cell layer, or outer nuclear or plexiform layers. These findings of enkephalin-like immunoreactive cells and fibers in a mammalian retina add to the findings in nonmammalian retinae and suggest that the enkephalins play a role in primary sensory systems of mammalian and nonmammalian species.
Leukemogenesis by bovine leukemia virus: proviral DNA integration and lack of RNA expression of viral long terminal repeat and 3' proximate cellular sequences.
Authors: Kettmann R , Deschamps J , Cleuter Y , Couez D , Burny A , Marbaix G
Published: May 2006

The DNA from 17 lymphoid tumors induced by bovine leukemia virus (BLV) was digested with the restriction endonuclease EcoRI. Filter hybridization analysis using radioactive probes specific for the BLV genome showed that all tumors contained at least one or a portion of one provirus. Digestion of these proviruses with Sac I demonstrated that deletions occurred in about 25% of the cases and involved sequences located in the 5' half of the provirus. No sequence homology was observed between the cloned proximate cellular sequences flanking two different proviruses at their 3' end and the corresponding sequences in 16 other tumor DNAs, thus showing that a wide range of genomic sites could accommodate BLV proviruses. Transcription of viral DNA including long terminal repeated sequences was not detected, strongly suggesting that viral gene expression is not required for maintenance of the tumor state. No expression of 3'-proximate cellular sequences was observed, indicating that no proximate downstream promotion took place in the cases examined.
Production in Escherichia coli of biologically active secretin, a gastrointestinal hormone.
Authors: Suzuki M , Sumi S , Hasegawa A , Nishizawa T , Miyoshi K , Wakisaka S , Miyake T , Misoka F
Published: May 2006

A synthetic gene for porcine secretin was ligated with Pst I-cleaved pBR322 by its flanking synthetic Pst I linkers to produce a fused protein consisting of an amino-terminal portion of beta-lactamase and an entire secretin molecule. The hybrid plasmid was transferred into competent Escherichia coli cells. The plasmids, which were proved in our investigation to contain the secretin gene in the desired orientation, were then screened for the production of secretin. This was shown by radioimmunoassay and gel electrophoretic analysis of the polypeptides that were synthesized in E. coli minicells transformed with the hybrid plasmids. Secretin so produced, whose carboxy terminal residue may not be amidated in contrast with natural porcine secretin, showed the same activity in stimulating pancreatic secretion in a bioassay with anesthetized rats as does natural secretin.
Monoclonal antibodies that recognize discrete forms of tubulin.
Authors: Gozes I , Barnstable C J
Published: May 2006

Anti-tubulin antibodies secreted by plasmacytoma NSI-spleen cell hybrids were detected by an indirect binding assay. Different antibodies bound to different combinations of the tubulins as resolved by isoelectric focusing. Two monoclonal antibodies (TUB 2.1 and TUB 2.5) labeled only (i) the tubulin band on a polyacrylamide electropherogram and (ii) beta-tubulins as resolved by isoelectric focusing. The fraction that was specifically bound and eluted from antibody affinity columns was enriched in beta-tubulins as compared with alpha-tubulins, suggesting the possibility of some soluble tubulin homodimers and alpha,beta-heterodimers. Double labeling experiments were used to show that all detectable microtubules contained beta-tubulin.
DNA sequence organization of IS
10
-right of Tn
10
and comparison with IS
10
-left
Authors: Halling S. M. , Simons R. W. , Way J. C. , Walsh R. B. , Kleckner N.
Published: May 2006

Tn
10
is 9,300 base pairs long and has inverted repeats of an insertion sequence (IS)-like sequence (IS
10
) at its ends. IS
10
-right provides all of the Tn
10
-encoded functions used for normal Tn
10
transposition. IS
10
-left can also provide these functions but at a much reduced level. We report here the complete nucleotide sequence of IS
10
-right and a partial sequence of IS
10
-left. From our analysis of this information, we draw the following conclusions. (
i
) IS
10
-right is 1,329 base pairs long. Like most IS elements, it has short (23-base pair) nearly perfect inverted repeats at its termini. We can divide these 23-base pair segments into at least two functionally distinct parts. IS
10
-right also shares with other elements the presence of a single long coding region that extends the entire length of the element. Genetic evidence suggests that this coding region specifies an essential IS
10
transposition function. A second, overlapping, coding region may or may not be important. (
ii
) The “outside” end of IS
10
-right contains three suggestively positioned internal symmetries. Two of these (A1 and A2) are nearly identical in sequence. Symmetry A1 overlaps the terminal inverted repeat; symmetry A2 overlaps the promoter shown elsewhere to be responsible for expression of IS
10
functions and lies very near a second characterized promoter that directs transcription outward across the end of IS
10
. Symmetries A1 and A2 may play a role in modulation of Tn
10
activity and are likely to function at least in part as protein recognition sites. We propose that the third symmetry (B) acts to prevent fortuitous expression of IS
10
functions from external promoters. The transcripts from such promoters can assume a stable secondary structure in which the AUG start codon of the long coding region is sequestered in a region of double-stranded mRNA formed by pairing between the two halves of symmetry B. (
iii
) IS
10
-left differs from IS
10
-right at many nucleotide positions in both the presumptive regulatory region and the long coding region. The available evidence suggests that Tn
10
may be older than other analyzed drug-resistance transposons and thus have had more time to accumulate mutational changes.
Cellular and subcellular localization of protein I in the peripheral nervous system.
Authors: Fried G , Nestler E J , De Camilli P , Stjärne L , Olson L , Lundberg J M , Hökfelt T , Ouimet C C , Greengard P
Published: May 2006

The cellular and subcellular distribution of protein I, a major brain phosphoprotein, has been studied in the peripheral nervous system. The levels of protein I in various peripheral nerves and innervated peripheral tissues were determined by radioimmunoassay and radioimmunolabeling of polyacrylamide gels. The results indicated tha protein I is present throughout the peripheral nervous system. Denervation studies of adrenal medulla and iris suggested that the protein I contained in peripheral tissues is localized to the neuronal elements innervating those tissues. Protein I was found to be enriched in neurotransmitter vesicle fractions of peripheral nervous tissue. Moreover, protein I appeared to be transported from cell bodies to axons terminals at least partly in association with neurotransmitter vesicles.
Identification and characterization of a proton pump on lysosomes by fluorescein-isothiocyanate-dextran fluorescence.
Authors: Ohkuma S , Moriyama Y , Takano T
Published: May 2006

Fluorescein isothiocyanate-conjugated dextran was introduced preferentially into hepatic lysosomes by intraperitoneal injection into rats. The pH in isolated lysosomes, measured by fluorescein fluorescence, was approximately 5 and gradually increased in KCl (to 7.0) at 25 degrees C. In the presence of Mg2+, ATP caused acidification of lysosomes that was reversed by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Mn2+, Co2+, and Fe2+ could replace Mg2+ but Ca2+ could not. Cu2+, Zn2+, and Cd2+ were inhibitory. A membrane-permeant anion, in practice chloride, was required for this acidification. ATP analogues, including 5'-adenylyl imidodiphosphate, could not be substituted for ATP. ATP-driven acidification was sensitive to N-ethylmaleimide and quercetin but insensitive to oligomycin, ouabain, and vanadate. There were some differences between "normal" lysosomes and tritosomes; the acidification was resistant to azide and N,N'-dicyclohexylcarbodiimide in normal lysosomes but sensitive to these reagents in tritosomes. These results provide evidence for the presence of an electrogenic proton pump driven by MgATP (H+-ATPase) on lysosomes.