bluuebunny/crossref_metadata_2025 huggingface dataset:
Biosynthesis of carbonic anhydrase in
Chlamydomonas reinhardtii
during adaptation to low CO
2
Authors: Coleman John R. , Grossman Arthur R.
Published: May 2006

The unicellular green alga
Chlamydomonas reinhardtii
synthesizes carbonic anhydrase in response to low levels of CO
2
(i.e., air levels of CO
2
). This enzyme, localized predominantly in the periplasmic space of the alga (or associated with the cell wall), is an important component of the machinery required for the active accumulation of inorganic carbon by
C. reinhardtii
and the saturation of ribulose-1,5-bisphosphate carboxylase at low extracellular carbon concentrations. We have begun to examine the synthesis and compartmentalization of carbonic anhydrase in
C. reinhardtii
. The monomeric species associated with carbonic anhydrase activity is synthesized as a precursor on 80S cytoplasmic ribosomes. This precursor can be detected immunologically in the profiles of translation products when a reticulocyte lysate, cell-free system is primed with poly(A)-RNA from either air-grown
C. reinhardtii
or cells shifted from growth on 5% CO
2
to air for 12 hr. It is not synthesized when the
in vitro
system is primed with poly(A)-RNA from CO
2
-grown algae. Since translatable RNA for the polypeptide responsible for carbonic anhydrase activity was only present in cells that experienced low levels of CO
2
, the adaptation process either involves the regulation of transcription of the carbonic anhydrase gene (and perhaps other genes involved in adaptation) or the post-transcriptional processing of the messenger RNA. Furthermore, the appearance of the mature polypeptide associated with carbonic anhydrase activity in the periplasmic space of
C. reinhardtii
is inhibited by tunicamycin, an antibiotic that prevents core glycosylation of polypeptides on the endoplasmic reticulum. Together, these results suggest that the biosynthesis of this extracellular algal enzyme involves the translation of mRNA for the carbonic anhydrase monomer on ribosomes bound to the endoplasmic reticulum, the cleavage of a signal sequence during transport of the nascent polypeptide into the lumen of the endoplasmic reticulum, and subsequent glycosylation events prior to export across the plasmalemma.
Evidence for physiological function of epidermal growth factor: pregestational sialoadenectomy of mice decreases milk production and increases offspring mortality during lactation period.
Authors: Okamoto S , Oka T
Published: May 2006

Female virgin mice, whose submandibular glands were removed, underwent normal pregnancy and delivery. During the nursing period, however, a substantial number of pups born to and nursed by sialoadenectomized mothers died within 5 days of birth, whereas this did not occur among pups born to normal mothers. Cross-foster nursing experiments indicated that the cause of death of pups was to be found in sialoadenectomized mothers, not in the pups. The capacity of the sialoadenectomized mothers to nurse pups was much less than that of normal mothers, as shown by experiments involving alterations in the number of pups nursed by both sialoadenectomized and normal mothers. The mammary gland of lactating sialoadenectomized mice was smaller in size and produced less milk compared with that of normal mice. No apparent qualitative difference in milk proteins was found in the milk produced by the two groups of mothers. The decreased growth of the mammary gland of sialoadenectomized mice was also manifested during the second half of pregnancy, and mammary explants from those mice synthesized less casein in response to lactogenic stimuli, insulin, cortisol, and prolactin, in an organ culture system, when compared with mammary explants from normal pregnant mice. When epidermal growth factor, a polypeptide hormone that is synthesized and secreted by the submandibular gland, was injected daily at a dose of 5 micrograms into sialoadenectomized pregnant mice, the survival rate of the pups nursed by their mothers increased to the value obtained with normal mothers. The results were discussed in terms of a possible role of the submandibular gland and epidermal growth factor in the development of the mammary gland.
Extension of bacteriophage lambda host range: selection, cloning, and characterization of a constitutive lambda receptor gene.
Authors: de Vries G E , Raymond C K , Ludwig R A
Published: May 2006

A set of plasmids has been constructed that carry a constitutive lamB gene (LamBc phenotype) from Escherichia coli and that confer functional phage lambda receptors to bacteria other than E. coli. This E. coli LamBc strain has been selected to escape both maltose-inducible and glucose-repressible control. Constitutivity results from an IS-3 insertion, carrying a mobile promoter, proximal to lamB. The LamBc DNA has been cloned into both broad and narrow host-range plasmids, and the resulting pTROY plasmids have been transferred to diverse bacteria. Both Salmonella typhimurium/pTROY and Klebsiella pneumoniae/pTROY strains efficiently adsorb phage lambda; Pseudomonas aeruginosa/pTROY strains do not. Introduction of a functional E. coli LamB protein into foreign bacterial will allow these bacteria carrying pTROY plasmids to be infected by phage lambda recombinant DNA libraries, phage lambda::Tn insertion mutagenesis vectors, and in vivo lambda-packaged cosmids.
Herpes simplex virus latency in isolated human neurons.
Authors: Wigdahl B , Smith C A , Traglia H M , Rapp F
Published: May 2006

Herpes simplex virus is most probably maintained in the ganglion neurons of the peripheral nervous system of humans in a latent form that can reactivate to produce recurrent disease. As an approximation of this cell-virus interaction, we have constructed a herpes simplex virus latency in vitro model system using human fetus sensory neurons as the host cell. Human fetus neurons were characterized as neuronal in origin by the detection of the neuropeptide substance P and the neuron-specific plasma membrane A2B5 antigen. Virus latency was established by blocking complete expression of the virus genome by treatment of infected human neurons with a combination of human leukocyte interferon and (E)-5-(2-bromovinyl)-2'-deoxyuridine for 7 days. After removal of inhibitors, virus latency was maintained for at least 9 days. This in vitro model will provide a system to analyze, in a primary human neuron, the state of the herpes simplex virus genome during establishment and maintenance of experimental latency.
Population differences in complexity of a learned skill are correlated with the brain space involved.
Authors: Canady R A , Kroodsma D E , Nottebohm F
Published: May 2006

The song of marsh wrens (Cistothorus palustris) is a learned trait passed on from generation to generation. Male marsh wrens from California and New York learn about 150 and 50 different songs apiece, respectively. The volumes of the hyperstriatum ventralis, pars caudalis and the robust nucleus of the archistriatum, two telencephalic nuclei involved in song control, are larger by an average of 40% and 30%, respectively, in the population with a larger song repertoire.
Xenopsin: the neurotensin-like octapeptide from Xenopus skin at the carboxyl terminus of its precursor.
Authors: Sures I , Crippa M
Published: May 2006

We have synthesized two oligodeoxyribonucleotide mixtures that contain sequences complementary to different parts of the hypothetical mRNA sequence of xenopsin, a biologically active octapeptide found in skin extracts from Xenopus laevis. The two primer pools were independently used to initiate reverse transcription on skin poly(A)+ RNA and the resulting cDNAs were then used to screen in parallel a cDNA library prepared from skin poly(A)+ RNA. One of the clones that hybridized with both probes was subjected to sequence analysis. It contains a nearly full-length DNA copy of a mRNA of approximately equal to 490 nucleotides that encodes a xenopsin precursor protein. The deduced precursor is 80 amino acids long, exhibits a putative signal sequence at the NH2 terminus, and contains the biologically active peptide at the COOH terminus. The region corresponding to the NH2-terminal portion of the xenopsin precursor shows a striking nucleotide and amino acid sequence homology with the precursor of PYLa, another recently described peptide from Xenopus skin.
Mutational analysis of a regulatory region in bacteriophage lambda that has overlapping signals for the initiation of transcription and translation.
Authors: Wulff D L , Mahoney M , Shatzman A , Rosenberg M
Published: May 2006

The positively regulated PRE promoter of phage lambda structurally overlaps with the ribosome-binding and NH2-terminal coding region of the regulatory protein (cII) that activates PRE transcription. We have isolated and characterized 27 different point mutations that occur within the 36-base-pair overlapping region. A comparison of genetic crossover data with nucleotide separations as determined by DNA sequence analysis reveals that recombination frequencies are greatly depressed at very short distances. Moreover, recombination frequency is critically dependent upon the precise nucleotide sequence of the crossover region for distances of five nucleotides or less. The mutations define precise positions and sequences that are important to (i) PRE promoter function, (ii) translation of the cII gene, and (iii) cII gene function. Mutational changes that affect the function of one element in this region concomitantly define phenotypically silent alterations in the other two elements. Mutations deficient in promoter function (P-RE or cy) are clustered in two regions that lie approximately equal to 10 and approximately equal to 35 nucleotides before the initial base of PRE mRNA, analogous to mutations in other promoters. P-RE mutations in the -10 region alter bases that are conserved in prokaryotic promoters, but P-RE mutations in the -35 region do not affect bases that are normally conserved in other promoters. Several mutations deficient in cII gene activity affect the initiation of cII protein synthesis, including an A leads to G change four bases outside the cII coding region, and AUG leads to GUG, AUG leads to ACG, and AUG leads to AUA mutations in the initiation codon. In the region of overlap between the PRE promoter and the NH2-terminal region of the cII gene, most amino acid substitutions in the cII protein do not result in a loss of cII function, indicating that this region of the gene does not contain essential information for cII function. We suggest that the overlap itself is an evolutionarily conserved structure and that it somehow coordinates the bidirectional transcriptional and translational events that occur in this region.
Neutron-capture therapy of human cancer: in vivo results on tumor localization of boron-10-labeled antibodies to carcinoembryonic antigen in the GW-39 tumor model system.
Authors: Goldenberg D M , Sharkey R M , Primus F J , Mizusawa E , Hawthorne M F
Published: May 2006

Antibody against carcinoembryonic antigen (CEA) was conjugated with p-[1,2-dicarba-closo-[1-3H]do-decaboran(12)-2-yl] benzenediazonium ion by an azo-coupling reaction, resulting in 30 boron atoms per IgG molecule with no loss of antibody protein. Antibody immunoreactivity was not appreciably affected by this conjugation and was stable after incubation in vitro in hamster plasma for 24 hr. The efficacy of the boron-conjugated anti-CEA IgG for localizing selectively in CEA-containing human colonic carcinomas propagated in the hind leg musculature of hamsters was evaluated by labeling the antibodies with 131I and determining distribution of the radioactivity in vivo. The results show that the boron-conjugated antibodies retain selective localization in the tumors, thus indicating their suitability for transporting boron-10 to tumors for use in neutron-capture therapy of cancer.
Cell surface antigens of human ovarian and endometrial carcinoma defined by mouse monoclonal antibodies.
Authors: Mattes M J , Cordon-Cardo C , Lewis J L , Old L J , Lloyd K O
Published: May 2006

Mouse monoclonal antibodies to several cell surface antigens of human ovarian and endometrial carcinomas have been produced. The distribution of the antigens was determined by mixed hemagglutination assays on 153 normal and malignant cell cultures of various types and by immuno-peroxidase staining of frozen sections of 27 normal adult and 24 fetal tissues. Five distinct antigens were characterized. MD144 antigen was detected on only a single ovarian carcinoma cell line and has the biochemical properties of a lipid. MH55 antigen is weakly expressed on ovarian and uterine cancer cell lines but not on other cells and tissues tested. MF61 antigen was detected on an ovarian carcinoma and some renal carcinoma cell lines but not on other cell lines tested. It was also detected by immunoperoxidase staining in the noncellular follicles of the thyroid and in uterine glandular epithelial cells. This antigen also has the properties of a lipid. MF116 antigen was detected on a proportion of ovarian, uterine, renal, and bladder carcinoma and neuroblastoma cell lines and on normal kidney epithelial cell cultures but not on other cell lines tested. It was not detected in sections of any normal tissue tested using the immunoperoxidase method. MF116 was readily detected in the spent culture medium but not in detergent-solubilized extracts of metabolically radiolabeled cells. This shed antigen is a glycoprotein of Mr 105,000 and isoelectric point lower than pH 4.0. MH94 antigen was detected on a proportion of ovarian, uterine, colon, breast, lung, cervical, and pancreatic carcinoma cell lines. In tissue sections it was detected in many but not all epithelia, predominantly in secretory epithelial cells. Antibody MH94 did not immunoprecipitate a detectable antigen.
A bombesin immunoreactive peptide in milk.
Authors: Jahnke G D , Lazarus L H
Published: May 2006

Immunoreactivity to the amphibian peptide bombesin was found in instant nonfat dry milk (ca. 0.7 ng/ml) and in the whey of whole or skim bovine milk (ca. 1.2 ng/ml) even after ultracentrifugation. The soluble immunoreactivity was associated with a peptide exhibiting the following characteristics: (i) parallel displacement in an immunoassay using an antiserum recognizing bombesin amino acid residues 5-8; (ii) separation from both gastrin-releasing peptide and amphibian bombesin by gel filtration--the approximate Mr was 3,200; (iii) denaturation in urea, reduction by dithiothreitol, and acetylation by iodoacetamide had no effect on its elution profile by gel-filtration chromatography and the aggregation of added bombesin to milk proteins or peptides was not observed; (iv) reversed-phase HPLC separated milk immunoreactivity from gastrin-releasing peptide and bombesin; (v) digestion by trypsin yielded a smaller immunoreactive peptide fragment, whereas nearly all immunoreactivity was lost by treatment with alpha-chymotrypsin; and (vi) the level of immunoreactivity was unaffected by boiling. These data show that milk is an exogenous source of bombesin-like immunoreactivity, which may account for the increase of gastric acid and gastrointestinal hormone levels after the consumption of milk.
Isolation and characterization of a cDNA clone for bovine cytochrome c oxidase subunit IV.
Authors: Lomax M I , Bachman N J , Nasoff M S , Caruthers M H , Grossman L I
Published: May 2006

We have isolated a cDNA clone for the precursor to subunit IV of bovine cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1). A cDNA library was constructed from poly(A)+ RNA of adult beef liver by insertion of cDNA into the plasmid vector pBR322. Transformants were screened by colony hybridization with two mixtures of [32P]-labeled synthetic oligodeoxyribonucleotides. We screened 20,000 transformants with a mixture of heptadecamers complementary to all 16 possible sequences encoding amino acids 98-103 and obtained two cDNA clones encoding subunit IV amino acid sequences. We determined the DNA sequence of the larger (416 base-pair) insert, which contains the coding sequence for amino acids 1-107 of the mature protein and an NH2-terminal extension (presequence). The deduced amino acid sequence of the mature protein is identical with the previously determined protein sequence: the sequence of the NH2-terminal extension contains a potential initiator methionine at amino acid -22 from the NH2-terminus of the processed protein. The presequence is quite basic and contains several arginines, including one at the processing site. No hydrophobic region analogous to that found in bacterial and eukaryotic signal peptides is present, but there are homologies with other mitochondrial protein presequences, which may include a common signal for their destination and processing.
Somatic cell genetic analysis of human cell surface antigens: chromosomal assignments and regulation of expression in rodent-human hybrid cells.
Authors: Rettig W J , Dracopoli N C , Goetzger T A , Spengler B A , Biedler J L , Oettgen H F , Old L J
Published: May 2006

The expression of 13 newly defined human cell surface antigens identified by monoclonal antibodies was studied in a panel of reduced rodent-human somatic cell hybrid clones. For each antigenic system the segregation of antibody reactivity was concordant with the segregation of a specific human chromosome, permitting the chromosomal assignment of 13 gene loci determining antigen expression. The antigens can be placed in four groups on the basis of their patterns of control in the hybrid cells. (i) Presence of a single human chromosome is necessary and sufficient for antigen expression; L230 (assigned to chromosome 2), AJ425, K15 (chromosome 3), SR84 (chromosome 5), JF23, Q14 (chromosome 11), SV13 (chromosome 15), and F10 (chromosome 19). (ii) AJ2 (chromosome 10) and J143 (chromosome 17); two antigens coded for by separate human chromosomes but associated as a molecular complex on the surface of AJ2+/J143+ human cells. (iii) F8 (chromosome 19); antigen expression dependent on the growth characteristics of hybrid cells: substrate-adherent cells are F8+, whereas cells growing in suspension are F8-. (iv) AO122 and F23 (chromosome 15); antigen expression controlled by the permissive/inducing vs. nonpermissive/noninducing nature of the rodent fusion partner. Hybrids derived from both antigen-positive and antigen-negative human cells can express AO122 and F23 but only when specific rodent cell types are used for hybridization: N4TG-1 neuroblastoma and L cells, but not RAG renal carcinoma cells, permit AO122 expression, whereas RAG and L cells, but not N4TG-1 cells, permit F23 expression. The rapidly expanding list of monoclonal antibodies defining human cell surface molecules provides a range of markers to probe the genetic regulation of antigen diversity in somatic cells.
Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells.
Authors: Hermonat P L , Muzyczka N
Published: May 2006

Adeno-associated virus (AAV) is a human DNA virus that has a broad host range and can be grown both as an integrated provirus and as a lytic genome. These properties suggested that AAV may be useful as a mammalian transduction vector. To test this possibility, we have isolated a recombinant AAV viral stock in which the neomycin resistance gene was substituted for the AAV capsid genes. Using this recombinant stock, we have demonstrated that AAV can be used to transduce foreign DNA into human and murine tissue culture cells. In addition, we have demonstrated that, if the transductants are superinfected with a helper virus (adenovirus), the recombinant AAV genome is rescued from the proviral state and amplified to high copy number. These unique features of AAV vectors suggest that they may have a broad utility in the study of biological problems. Because AAV, itself, is nonpathogenic in both humans and animals, these vectors also may be useful for the purpose of gene therapy.
Detection and immunochemical characterization of a primate type C retrovirus-related p30 protein in normal human placentas.
Authors: Jerabek L B , Mellors R C , Elkon K B , Mellors J W
Published: May 2006

We find that 12 of 14 specimens of normal human term placentas analyzed by one- or two-dimensional electrophoresis and immunoblotting contain a protein or polypeptide of approximately equal to 30,000 daltons that is antigenically cross-reactive with p30 core protein of the simian sarcoma-associated virus/gibbon ape leukemia virus primate retrovirus group and is physicochemically similar to reference murine and primate type C retrovirus p30s. This finding may lead to an understanding of endogenous type C retrovirus gene expression in humans.
Preparation and characterization of pentaammineruthenium-(histidine-83)azurin: thermodynamics of intramolecular electron transfer from ruthenium to copper.
Authors: Margalit R , Kostić N M , Che C M , Blair D F , Chiang H J , Pecht I , Shelton J B , Shelton J R , Schroeder W A , Gray H B
Published: May 2006

The reaction between a5RuH2O2+ (a is NH3) and Pseudomonas aeruginosa azurin at pH 7, followed by oxidation, yields a5Ru(His-83)3+-azurin(Cu2+) as the major product. Spectroscopic measurements (UV-visible, CD, EPR, and resonance Raman) indicate that the native structure is maintained in the modified protein. The site of ruthenium binding (His-83) was identified by peptide mapping. The a5RuHis/Cu ratio in the modified protein, determined from the EPR spectrum, is 1:1, and the reduction potentials (vs. normal hydrogen electrode, pH 7.0, 25 degrees C) are blue copper (Cu2+/1+), 320 +/- 2 mV; a5Ru(His-83)3+/2+, 50 +/- 10 mV. From measurements of the reduction potentials at several temperatures in the 5-40 degrees C range, delta H degree for intramolecular Ru2+ ----Cu2+ electron transfer was estimated to be -12.4 kcal mol-1 (1 cal = 4.184 J). Analysis of kinetic data in light of the electron transfer exothermicity indicates that the reorganizational enthalpy of the blue copper site can be no larger than 7.1 kcal mol-1.
Monomeric solubilized sarcoplasmic reticulum Ca pump protein: demonstration of Ca binding and dissociation coupled to ATP hydrolysis.
Authors: Martin D W , Tanford C , Reynolds J A
Published: May 2006

The sarcoplasmic reticulum Ca-pump protein can be solubilized in monomeric form in nonionic detergents with full retention of ATPase activity. It is impossible to prove directly that coupling between ATP hydrolysis and Ca2+ transport is maintained in the soluble state, because separate compartments for Ca2+ uptake and Ca2+ discharge are required to demonstrate this, but here we provide strong indirect evidence that coupling in fact persists, in both the forward and reverse directions of the normal pump cycle. Demonstration of coupling in the forward direction makes use of the fact that the solubilized protein is structurally labile in the absence of bound Ca2+. Loss of activity accompanying ATP hydrolysis in solution is quantitatively consistent with sequential Ca2+ binding and dissociation during the hydrolysis cycle. Coupling in the reverse direction is demonstrated by the dependence of ATP synthesis on Ca2+ concentration. Although substantial differences between solubilized (monomeric) and membrane-bound protein are shown to exist, as previously reported, they do not affect the main conclusion from this work, which is that the molecular machinery for free-energy coupling in active Ca2+ transport is an inherent property of each individual catalytic polypeptide chain of the pump protein.
Integration-specific retrovirus expression in embryonal carcinoma cells.
Authors: Sorge J , Cutting A E , Erdman V D , Gautsch J W
Published: May 2006

The undifferentiated embryonal carcinoma (EC) cell line PCC4 aza 1 was infected with a selectable amphotropic retrovirus. Although EC cells are generally refractory to retroviral gene expression, we found that approximately 1 of 5,000-10,000 recombinant proviruses was transcribed in these cells. Cells containing an active recombinant provirus were cloned and expanded. Nucleic acid analysis revealed approximately one intact provirus per cell. Viral RNA levels were different for each cell clone examined [between 0.05% and 0.5% of the poly(A)+ RNA], and transcription was initiated and terminated in the long terminal repeats as in permissive differentiated cells. The cells retained an undifferentiated phenotype and remained positive for stage-specific embryonic antigen 1 and negative for H-2 surface antigens. The cells retained the block to the expression of other proviruses integrated at other chromosomal locations. The data suggest that a cis-acting genetic element, at or near the chromosomal site of integration, or mutations in the viral control elements themselves may be responsible for provirus expression in these cells.
Target polypeptide of a carcinogen is associated with normal mitosis and carcinogen-induced hyperplasias in adult hepatocytes.
Authors: Custer R P , Sorof S
Published: May 2006

Normal rat liver cytosol was found previously to contain a 14,000-dalton polypeptide that is the principal target of the carcinogen N-2-fluorenylacetamide (2-acetyl-aminofluorene) early during hepatocarcinogenesis. By using antiserum that identifies the 14,000-dalton polypeptide in liver cytosol, an immunologically related 17,500-dalton polypeptide was shown to be present in isolated normal liver nuclei, tightly bound to chromatin. We report here that the 14,000-dalton polypeptide is associated with cell multiplication in normal adult hepatocytes. Of 150 hepatocytes in five stages of mitosis, all invariably displayed a greatly increased concentration of the 14,000-dalton polypeptide in cytoplasm, compared to hepatocytes in interphase, by immunostaining with peroxidase-antiperoxidase complex. In contrast, mitosis did not appear to affect the level of the 17,500-dalton polypeptide in nuclei. A small population of high-polyploid hepatocytes with large nuclei had elevated intensities of cytoplasmic immunostain that approached that in mitotic cells. The increased level of discernible 14,000-dalton polypeptide target of the carcinogen in cytoplasm is a marker of the rare mitotic hepatocytes in normal adult rat liver. Ingestion of the liver carcinogen N-2-fluorenylacetamide for 5 wk or an azocarcinogen, 3'-methyl-4-dimethylaminoazobenzene, for 4 wk brought about early foci of hyperplastic hepatocytes in which there was a great overload of detectable 14,000-dalton target polypeptide in their cytoplasm and a near absence of the 17,500-dalton polypeptide in most nuclei. In contrast, the cytoplasm and nuclei in surrounding morphologically nontransformed hepatocytes of livers of the carcinogen-fed rats immunostained to a much smaller degree than in normal adult hepatocytes. Removal of the azo-dye carcinogen for 4 wk resulted in the disappearance of most hyperplastic foci. A few foci did persist and had similar apparent abnormal levels of the two polypeptides. The high concentration of discernible 14,000-dalton target polypeptide in the cytoplasm of hepatocytes in the hyperplastic foci correlates with their known proliferation, whereas the low cytoplasmic level in the surrounding hepatocytes is consistent with the known inhibition of their mitosis by these carcinogens. The collected evidence appears to joint together a chemical carcinogen, a cytoplasmic principal target polypeptide, a chromatin-bound polypeptide, mitosis in normal adult hepatocytes, early and persistent hyperplastic foci caused by carcinogens, and the inhibition of mitosis by carcinogens in surrounding parenchyma in preneoplastic livers.
Differential processing of prodynorphin and proenkephalin in specific regions of the rat brain.
Authors: Zamir N , Weber E , Palkovits M , Brownstein M
Published: May 2006

Prodynorphin-derived peptides [dynorphin A (Dyn A)-(1-17), Dyn A-(1-8), Dyn B, alpha-neo-endorphin, and beta-neo-endorphin] and proenkephalin-derived peptides [[Leu]enkephalin [( Leu]Enk) and [Met]enkephalin-Arg6-Gly7-Leu8 [( Met]Enk-Arg-Gly-Leu]) in selected brain areas of the rat were measured by specific radioimmunoassays. We report here that different regions of rat brain contain strikingly different proportions of the prodynorphin and proenkephalin-derived peptides. There is a molar excess of alpha-neo-endorphin-derived peptides over Dyn B and Dyn A-derived peptides in many brain areas. [Leu]Enk concentrations exceed those of [Met]Enk-Arg-Gly-Leu in certain brain areas such as the substantia nigra, dentate gyrus, globus pallidus, and median eminence (areas rich in dynorphin-related peptides). These results indicated that (i) there is differential processing of prodynorphin in different brain regions and (ii) [Leu]Enk may be derived from Dyn A or Dyn B (or both). In certain brain regions [Leu]Enk may derive from two separate precursors (prodynorphin and proenkephalin) in two distinct neuronal systems.
Isolation and characterization of a cDNA clone for the gamma subunit of bovine retinal transducin.
Authors: Hurley J B , Fong H K , Teplow D B , Dreyer W J , Simon M I
Published: May 2006

We have isolated and characterized a cDNA clone that encodes the gamma subunit of transducin, the guanine nucleotide binding regulatory protein found in vertebrate photoreceptors. The gamma subunit was separated from the alpha and beta subunits of transducin and purified to homogeneity by reversed-phase high performance liquid chromatography. The sequence of the first 45 amino acids at the amino terminus of this polypeptide was then determined by automated Edman degradation. Oligodeoxynucleotide probes corresponding to two nonoverlapping regions of this sequence were synthesized and then used to screen a bovine retinal cDNA library. One probe, T gamma 1, was a mixture of 32 different heptadecamers complementary to all possible mRNA sequences that could encode a portion of the T gamma sequence; the other probe, T gamma 2, was a mixture of 128 different heptadecamers. Thirteen clones that hybridized with T gamma 1 were selected. Only one of these had an insert that also hybridized with T gamma 2. The DNA sequence of this insert encodes a 73-amino acid polypeptide that corresponds to the transducin gamma subunit on the basis of amino-terminal sequence, amino acid composition, and carboxyl-terminal sequence. The molecular weight of the mature gamma subunit is 8400. It appears to be synthesized as a discrete polypeptide and not as a domain of a larger precursor polyprotein. The transducin gamma subunit is very hydrophilic and acidic; it has 19 acidic and 11 basic amino acids as well as three cysteine residues. Furthermore, significant homology was found in comparisons of the nucleic acid sequence corresponding to the carboxyl terminus of the gamma transducin transcript with the sequences corresponding to the carboxyl terminus of ras oncogene products, suggesting a possible ancestral relationship between these genes.
Guanosine nucleotide binding by highly purified Ha-ras-encoded p21 protein produced in Escherichia coli.
Authors: Manne V , Yamazaki S , Kung H F
Published: May 2006

High-level expression of the p21 protein product of the BALB murine sarcoma virus v-ras gene (similar to the product of the Harvey murine sarcoma virus v-Ha-ras gene) has been reported recently, and highly purified preparations of this protein have been obtained. We used a nitrocellulose filter assay for measuring the binding of GDP and GTP to the purified protein. Previously p21 antibodies had been used to precipitate p21-guanosine nucleotide complexes from crude extracts containing the protein. Using the filter assay, we find that the v-Ha-ras gene product binds [3H]GDP stoichiometrically. The binding is time-dependent and is faster at 30 degrees C than at 0 degrees C. Optimum binding is obtained in the presence of dithiothreitol and magnesium ions and at pH 7.4. In terms of its GDP binding activity, p21 is heat stable and pronase sensitive. The dissociation constants (Kd) of p21 for [3H]GDP and [3H]GTP, determined by Scatchard analysis, are 6 X 10(-8) M and 2.5 X 10(-8) M, respectively.
Induction of murine teratocarcinoma cell differentiation by suppression of poly(ADP-ribose) synthesis.
Authors: Ohashi Y , Ueda K , Hayaishi O , Ikai K , Niwa O
Published: May 2006

Poly(ADP-ribose) synthesizing activity in mouse teratocarcinoma EC-A1 cells decreased markedly during differentiation induced by retinoic acid; the activities assayed in permeabilized cells decreased to 25% and 10% of the activity of control (uninduced cells) 2 and 3 days, respectively, after the addition of 0.1 microM retinoic acid to the culture medium. This change preceded changes in morphology and DNA synthesis, which became prominent after 4 days. The decrease in poly(ADP-ribose) synthesizing activity appeared to be caused by a diminution of the synthetase protein and not by a decrease in its catalytic activity, because the full activity disclosed by DNase I treatment decreased in parallel, albeit at about 20 times higher levels. When 8 mM 3-aminobenzamide or 10 mM nicotinamide, specific inhibitors of poly(ADP-ribose) synthetase, was added to the culture medium, the cells underwent differentiation after 7-9 days. An analogue, 3-aminobenzoic acid, which is not inhibitory to the synthetase, induced differentiation much less efficiently than did 3-aminobenzamide, and the effect of 3-aminobenzoic acid appeared to be ascribable to its potent cytotoxicity. Immunohistochemical analysis using anti-poly(ADP-ribose) antibody confirmed the marked reduction in poly(ADP-ribose) synthesizing activity in nuclei of the cells treated with retinoic acid or 3-aminobenzamide but not with 3-aminobenzoic acid. These results suggest that a decrease in poly(ADP-ribose) synthesis triggers differentiation of teratocarcinoma cells.
Human U1 RNA genes contain an unusually sensitive nuclease S1 cleavage site within the conserved 3' flanking region.
Authors: Htun H , Lund E , Dahlberg J E
Published: May 2006

We find that the cloned DNAs of human U1 small nuclear RNA genes contain two nuclease S1-sensitive sites, one about 1.8 kilobases downstream of the U1 RNA coding region and the other around 0.3 kilobase upstream. The downstream site is unusually sensitive to the nuclease, being cleaved in both linear and negatively supercoiled DNAs. The extent of cleavage at this site is enhanced at lower pH and reduced concentrations of NaCl; the effects of salt are more apparent on linear than supercoiled DNAs. The nuclease S1 sensitivity of this downstream site is dependent on the presence of the sequence (dC-dT)n X (dA-dG)n, where n = 15-25. (One gene with n = 5 is resistant to nuclease S1 cleavage in this region.) In contrast, the nuclease S1 site upstream of the coding region is cleaved only when the DNA is supercoiled. This site also has a homopyrimidine X homopurine bias in the DNA strands, but the sequence is less regular. In the course of these studies, we detected several discrepancies between our restriction maps of some U1 RNA genes and those published by others. Our maps demonstrate that all seven cloned human U1 RNA genes are very similar in sequence for as much as 2.3 kilobases downstream of the U1 RNA coding region.
Avidin-biotin affinity chromatography: application to the isolation of human placental insulin receptor.
Authors: Finn F M , Titus G , Horstman D , Hofmann K
Published: May 2006

The ligand N alpha, B1-(6-biotinylamido)hexanoyl-insulin was attached noncovalently to Sepharose 4B immobilized succinoylavidin to form an insulin-affinity resin. This resin was used to isolate highly purified insulin receptor from human placental tissue by a four step process involving (i) preparation of a crude membrane fraction, (ii) solubilization with Triton X-100, (iii) wheat germ agglutinin purification, and (iv) insulin-affinity chromatography. NaDodSO4/PAGE of the purified 125I-labeled receptor under nonreducing conditions showed the presence of a major component with an approximate molecular weight of 350,000 and a minor component with a molecular weight of approximately equal to 166,000. Based on the assumption that the degree of labeling is comparable in both components, the material corresponding to the Mr 350,000 peak represents approximately equal to 94% of the receptor preparation as determined by scanning the autoradiograms. The specific insulin binding capacity of the preparation is 18 +/- 6 micrograms of 125I-labeled insulin per mg of protein as determined by the polyethylene glycol assay and analyzed by Scatchard plot. Insulin binding activity was stable at 4 degrees C and pH 7.6 for at least 12 weeks but was destroyed by freezing and thawing. The availability of highly purified receptor afforded the opportunity to explore its precipitability by polyethylene glycol under assay conditions. Whereas trichloroacetic acid precipitated 95% of the 125I-labeled receptor, polyethylene glycol precipitated only 30%. If the specific activity of the receptor is corrected for incomplete precipitability by polyethylene glycol, the apparent specific binding would be 3.5 +/- 1.2 mol of insulin per mol of receptor. These results are in disagreement with the current receptor model, which postulates that 1 mol of receptor (Mr, 350,000) binds 2 mol of insulin. Clearly, the problems associated with the method available for determining insulin binding are sufficiently serious to preclude their use in determining receptor valence.
Monoclonal antibodies to human type IV collagen: useful reagents to demonstrate the heterotrimeric nature of the molecule.
Authors: Odermatt B F , Lang A B , Rüttner J R , Winterhalter K H , Trüeb B
Published: May 2006

Monoclonal antibodies (mAbs) have been prepared against type IV collagen isolated from human kidney. Two mAbs, designated CIV 22 and CIV 16, were extensively characterized. CIV 22 reacted only with native type IV collagen, whereas CIV 16 also bound to fragments derived from the alpha 1(IV) chain after reduction and alkylation of the molecule. Therefore, CIV 22 recognizes a conformational epitope on the triple helical type IV collagen, whereas CIV 16 binds to a sequential determinant in the carboxyl-terminal half of the alpha 1(IV) chain. By immunofluorescence, typical basement membrane structures were stained with both mAbs on frozen sections of different human organs. The mAbs were used to investigate the chain composition of type IV collagen. Radiolabeled type IV collagen bound to CIV 22, proving its triple helical configuration. These native probes, containing both the alpha 1(IV) and the alpha 2(IV) chains, also bound to CIV 16. Since CIV 16 does not react with the isolated alpha 2(IV) chain, both chains must be arranged in a single triple helical molecule (heterotrimer).
Structural genes of dinitrogenase and dinitrogenase reductase are transcribed from two separate promoters in the broad host range cowpea
Rhizobium
strain IRc78
Authors: Yun Allen C. , Szalay Aladar A.
Published: May 2006

The nucleotide sequence of the structural gene (
nifD
) coding for the α-subunit of dinitrogenase along with its flanking sequences has been determined in cowpea
Rhizobium
IRc78. The coding sequence consists of 1500 nucleotides, which corresponds to a predicted amino acid sequence of 500 residues and a molecular weight of 56,025. Nucleotide homology to
nifD
from the blue-green alga,
Anabaena
, and
Parasponia Rhizobium
, are 63% and 90%, respectively. Cowpea
Rhizobium
IRc78
nifD
and
nifK
(encodes the β-subunit of dinitrogenase) genes are linked, separated by 69 nucleotides. In contrast to fast-growing rhizobia, the structural genes of dinitrogenase (
nifDK
) are transcribed from a different promoter than the structural gene of dinitrogenase reductase (
nifH
). Transcription of
nifDK
initiates 41 nucleotides upstream of the start codon for the
nifDK
operon. Two transcription initiation sites, localized at 152 and 114 nucleotides upstream of the start codon, were determined for the
nifH
operon. Two nucleotide sequences, a hexamer (G-G-T-T-G-C) and a pentamer (T-G-G-C-A), centered at approximately -15 and -25, respectively, are conserved in the
nifD
and
nifH
promoter regions and are not present in the 69-nucleotide
nifDK
junction. No sequence homology other than a possible ribosome binding site, T-T-G-A-[unk]-G-G-A, located 14 nucleotides upstream of the initiation codon was detected between the transcribed but untranslated leader regions of
nifD
and
nifH
.
Multiple initiation sites of DNA replication flanking the origin region of lambda dv genome.
Authors: Tsurimoto T , Matsubara K
Published: May 2006

Early replicative intermediates of lambda dv plasmid were prepared by an in vitro replication system in the presence of 2',3'-dideoxycytidine 5'-triphosphate, an inhibitor of DNA chain elongation. Short-chain DNAs produced from regions near the replication origin were purified from the intermediates. A fraction of the DNAs was covalently linked to primer RNA. The transition sites from primer RNA to DNA synthesis were mapped along the nucleotide sequence of the genome, by eliminating the RNA by alkaline hydrolysis and labeling the freshly exposed 5' ends of DNA with 32P. The transition sites were found to be located on both sides of the ori region, which includes four 19-base-pair repeats where one of the lambda specific initiator proteins, O, binds. No transition arose within the ori region. The transition sites are multiple on both sides of the ori region and are clustered in one of the two strands in such a way that DNA syntheses from the two sides converge. The frequency of the "leftward" DNA synthesis is several times higher than that of "rightward" synthesis, reflecting the asymmetric bidirectional replication of lambda dv DNA.
Intron splicing: a conserved internal signal in introns of animal pre-mRNAs.
Authors: Keller E B , Noon W A
Published: May 2006

Splicing of introns of yeast pre-mRNAs requires an internal conserved sequence T-A-C-T-A-A-C that is located 20-55 nucleotides from the 3' intron boundary. Sequences differing only in certain positions from this yeast signal have now been identified in the corresponding internal region of pre-mRNA introns of a variety of animal genes. A computer program that searches for homologues to a consensus structure and calculates the accuracy of match of each homologue is used to locate these sequences. We list here the signals found by this search in introns of sea urchin, mouse, rat, and human genes and give the consensus for each species. We also give the consensus found for Drosophila and chicken and duck signals. We then discuss the accumulating evidence that these internal signals are required for splicing in animals. It is also noted that a single-stranded region of small nuclear RNA U2 contains sequences complementary both to the proposed mammalian internal signal and to the neighboring CT-A-G at the 3' intron boundary. A role for U2 ribonucleoprotein in intron splicing is thus suggested.
myo-Inositol 1,4,5-trisphosphate stimulates protein phosphorylation in saponin-permeabilized human platelets.
Authors: Lapetina E G , Watson S P , Cuatrecasas P
Published: May 2006

In an attempt to establish a system with physiological substrates and phospholipid surfaces to investigate Ca2+- and 1,2-diacylglycerol-dependent protein kinase C activation, saponized platelets were used. Saponin, through interaction with plasma membrane cholesterol, makes cells permeable without major disruption of organelles. Washed platelets, prelabeled with 32P, were treated with 1-50 micrograms of saponin per ml. Permeabilization was evident at a concentration of 10 micrograms of saponin per ml, as indicated by the action of extracellular Ca2+ on the phosphorylation of the 20,000- and 40,000-Da proteins. These proteins are, respectively, the substrates for myosin light chain kinase and protein kinase C. Activation of these enzymes occurred when the estimated free [Ca2+] was changed from approximately equal to 80 nM to 300 nM. The effect of Ca2+ on kinase C-induced phosphorylation was potentiated by 1,2-didecanoylglycerol (1 microM). myo-Inositol 1,4,5-trisphosphate (5-20 microM) increased phosphorylation of the 20,000- and 40,000-Da proteins. This action was time and concentration dependent. The effect of myo-inositol 1,4,5-trisphosphate on the activation of kinase C was additive with 1,2-didecanoylglycerol. The action of myo-inositol 1,4,5-trisphosphate could be due to mobilization of Ca2+ from platelet organelles and/or to a direct effect on protein kinases.
Recognition of HLA-A2 and -B7 antigens by cloned cytotoxic T lymphocytes after gene transfer into human and monkey, but not mouse, cells.
Authors: Barbosa J A , Mentzer S J , Minowada G , Strominger J L , Burakoff S J , Biro P A
Published: May 2006

The genes that code for the human major histocompatibility class I antigens, HLA-A2 and HLA-B7, were introduced into human, monkey, and mouse cell lines by cotransfection with suitable biochemical markers and the fluorescence-activated cell sorter was used to identify and/or select stable cell populations expressing high surface levels of these antigens. Levels of expression obtained were similar to those observed for endogenous HLA antigens on various human cell lines and were 25-80% of those observed on the human B-lymphoblastoid cell line JY. Serologically defined HLA-A2 and HLA-B7 polymorphic determinants remained intact on all transfected recipient cells analyzed. Cloned human allospecific cytotoxic T lymphocytes (CTL) specific for HLA-A2 or HLA-B7 were capable of lysing appropriate HLA-transfected human cells with comparable efficiency to JY cell lysis. Two of 10 CTL clones lysed appropriate monkey cell transfectants with approximately equal to 20% the efficiency of human cell transfectants. No specific lysis of any HLA-transfected mouse cell lines, including a B cell lymphoma, was observed despite comparable levels of surface antigen expression or after induction of higher levels by mouse gamma-interferon. Furthermore, L cells expressing human beta 2-microglobulin in addition to HLA-A2 or -B7 were not lysed by these CTL. Thus, an additional species-specific component may be involved in lysis by allogeneic CTL--possibly related to the function(s) of other surface proteins on target cells.
Molecular cloning and sequence determination of rat preproenkephalin cDNA: sensitive probe for studying transcriptional changes in rat tissues.
Authors: Howells R D , Kilpatrick D L , Bhatt R , Monahan J J , Poonian M , Udenfriend S
Published: May 2006

A cDNA probe was prepared to investigate the regulation of proenkephalin biosynthesis in the rat. This was necessary because human and bovine proenkephalin cDNA were not sensitive enough for the accurate detection of preproenkephalin mRNA in tissues that contain low copy numbers of this message, such as the adrenal gland. The rat probe was prepared in the following manner. Preproenkephalin mRNA was enriched by sucrose gradient centrifugation of poly(A)-containing mRNA from rat brain and was used as a template for double-stranded cDNA synthesis. The resulting cDNA was inserted into the plasmid pBR322, and recombinant plasmids were used to transform Escherichia coli RR1 cells. A synthetic oligodeoxyribonucleotide (30 bases long) with a sequence that had previously been shown to be identical in bovine and human preproenkephalin cDNA was prepared to screen the clone bank. The plasmid with the longest cDNA insert (about 1200 bases) from the positive clones was isolated, and the sequence of the entire protein coding region was determined. Like the bovine and human gene products, rat preproenkephalin contains four [Met]enkephalin sequences and one copy each of [Leu]enkephalin, [Met]enkephalin-Arg6-Gly7-Leu8, and [Met]enkephalin-Arg6-Phe7. Rat preproenkephalin is 80% and 83% homologous to the bovine and human forms, respectively, at the nucleotide level and is 82% homologous to both species at the amino acid level. Rat preproenkephalin contains 269 amino acid residues, making it larger than the human (267 residues) and bovine (263 residues) precursors. The sensitivity for detection of rat preproenkephalin mRNA with the rat cDNA was several times greater than with the corresponding cDNAs from bovine and human sources.