bluuebunny/crossref_metadata_2025 huggingface dataset:
umuDC and mucAB operons whose products are required for UV light- and chemical-induced mutagenesis: UmuD, MucA, and LexA proteins share homology.
Authors: Perry K L , Elledge S J , Mitchell B B , Marsh L , Walker G C
Published: May 2006

The products of the Escherichia coli umuDC operon and its plasmid-borne analog, mucAB, are required for mutagenesis caused by UV light and by many chemicals. We have determined the nucleotide sequences of umuDC and mucAB and present comparisons of these sequences. The two operons are 52% homologous at the nucleotide level. Open reading frames corresponding in position and size to the umu and muc genes have been identified. The reading frames of umuD and umuC overlap by 1 base pair, and the reading frames of mucA and mucB overlap by 13 base pairs. The predicted amino acid sequences of the UmuD and MucA proteins are 41% homologous; those of the UmuC and MucB proteins are 55% homologous. Considerable homology has also been detected between UmuD, MucA, and the COOH-terminal domains of the LexA repressor and the repressors of phage lambda, 434, and P22. Complementation analyses reveal that MucA protein cannot substitute for UmuD in a umuD- umuC+ host and that MucB protein cannot substitute for UmuC in a umuD+ umuC- host. Potential regulatory sequences have been identified in umuDC and mucAB.
Transport to cell surface of intestinal sucrase-isomaltase is blocked in the Golgi apparatus in a patient with congenital sucrase-isomaltase deficiency.
Authors: Hauri H P , Roth J , Sterchi E E , Lentze M J
Published: May 2006

A case of congenital sucrase-isomaltase deficiency in man was investigated. An intestinal biopsy sample from a 5-year-old girl lacked sucrase but possessed low residual isomaltase activity. Immunoelectron microscopy with monoclonal antibodies to sucrase-isomaltase in biopsy samples from healthy subjects revealed that sucrase-isomaltase was confined predominantly to the microvillus membrane of enterocytes and there was minimal labeling of the Golgi apparatus. In the patient immunoreactive sucrase-isomaltase was found almost exclusively in about three trans-Golgi cisternae and associated vesicular structures, while no specific labeling was associated with the microvillus membrane. Immunoprecipitation experiments with iodinated mucosal homogenates and a mixture of four monoclonal antibodies to sucrase-isomaltase revealed absence of enzyme subunits in the patients but presence of a Mr 210,000 protein that was also expressed in normal control biopsy specimens. This protein presumably is the high-mannose precursor of sucrase-isomaltase. Additional proteins of Mr 160,000-200,000 found in the patient but not in normal subjects might correspond to the crossreacting material found in the Golgi apparatus of the patient. Overall, the findings suggest that in the patient sucrase-isomaltase is synthesized and transported to the Golgi apparatus, where further transport is interrupted. The data imply that signals in sucrase-isomaltase that mediate its transfer from the endoplasmic reticulum to the Golgi apparatus differ from those mediating its transport from the Golgi apparatus to the cell surface.
Lack of lymphocyte-induced DNA fragmentation in human targets during lysis represents a species-specific difference between human and murine cells.
Authors: Christiaansen J E , Sears D W
Published: May 2006

Significant cytotoxic lymphocyte-induced DNA fragmentation does not occur in four human target cells lysed by human natural killer lymphocytes, killer lymphocytes, or cytotoxic T lymphocytes. These results contrast with the extensive DNA fragmentation that occurs in murine target cells lysed by the analogous effector lymphocytes. Thus, DNA fragmentation, or the lack thereof, represents a species-specific difference between human and murine cells responding to lysis by cytotoxic lymphocytes. The DNase activity observed in murine cells is probably internally activated rather than delivered by the cytotoxic effector cells, since human killer lymphocytes selectively caused DNA fragmentation in murine but not in human target cells lysed by antibody-dependent cellular cytotoxicity.
Identification of genes for the constant region of rabbit T-cell receptor beta chains.
Authors: Angiolillo A L , Lamoyi E , Bernstein K E , Mage R G
Published: May 2006

We describe cDNA clones from thymus mRNA of a young rabbit that have sequences highly homologous to the human and murine T-cell receptor beta-chain constant region (C beta). In rabbit, man, and mouse there is a conserved extra cysteine in the constant region that could lead to a free thiol group or alternative disulfide bond formation depending on the locations and total numbers of cysteines in assembled receptor molecules. A cDNA clone (CL ANA 11) with 571 bases 5' of the C beta coding sequence has an open reading frame starting at a methionine codon that encodes 141 amino acids in frame with the C beta sequence. The encoded sequence has no resemblance to known immunoglobulin or beta-chain variable regions or other known proteins. An oligonucleotide probe from the 5' end of the encoded protein hybridizes to an approximately equal to 2-kilobase genomic DNA fragment that contains C beta gene sequences and to an approximately equal to 8-kilobase mRNA species in the thymus mRNA preparation from which the clone was derived. Within the 5' coding sequence there is a stretch of 211 bases containing strings of alternating purines and pyrimidines that may form Z-DNA. The sequence of the last 55 base pairs adjacent to C beta resembles the corresponding segment of mouse cDNA clone 86T3 that contains sequence 5' of the mouse C beta 1 gene. Although the function of a potential protein encoded by the 5' end of CL ANA 11 is unknown, it could play a role in regulation of thymocyte growth and differentiation.
Deficiency of electron transfer flavoprotein or electron transfer flavoprotein:ubiquinone oxidoreductase in glutaric acidemia type II fibroblasts.
Authors: Frerman F E , Goodman S I
Published: May 2006

Glutaric acidemia type II (GA II) is a human genetic disorder. It has been suggested that the primary defect in this disorder is a deficiency of a protein involved in electron transport between the acyl-CoA dehydrogenases and the bc1 complex of the mitochondrial respiratory chain. Antisera were raised to purified porcine electron transfer flavoprotein (ETF) and electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). The antisera were used to detect the two electron transferases in control and GA II fibroblasts by immunoblotting. Fibroblasts from three unrelated GA II patients were deficient in immunologically detectable ETF:QO and extracts from these three fibroblast lines contained no detectable ETF:QO catalytic activity. Fibroblasts from parents of two of these patients had ETF:QO activity intermediate between activities in control fibroblasts and fibroblasts from the patients. These data indicate that the primary defect in these patients is a deficiency of ETF:QO and that the mode of transmission of the gene is autosomal recessive. Fibroblasts from two other patients with severe GA II had normal levels of ETF-QO activity and antigen but were deficient in immunoreactive ETF. These findings show that GA II results from a deficiency of ETF in some patients and ETF:QO in others. In addition, these investigations provide strong evidence for the specificity and physiological function of the iron-sulfur flavoprotein ETF:QO.
Anatomical correlates of the distribution of the pathological changes in the neocortex in Alzheimer disease.
Authors: Pearson R C , Esiri M M , Hiorns R W , Wilcock G K , Powell T P
Published: May 2006

The numbers and distribution of the neurofibrillary tangles and neuritic plaques have been determined in several areas of the neocortex in brains affected by various degrees of severity of Alzheimer disease. The homotypical cortex of the "association" areas of the temporal, parietal, and frontal lobes are severely involved, whereas the motor, somatic sensory, and primary visual areas are virtually unaffected. The neurofibrillary tangles are mainly in the supra- and infragranular layers, particularly in layers III and V. In all areas except area 18 in the occipital lobe, there are approximately twice as many tangles in layer V as in layer III. The tangles are arranged in definite clusters, and those in the supra- and infragranular layers are in register. The neuritic plaques occur in all layers but predominantly affect layers II and III and do not show clustering. These data on the severity of the pathological involvement in different areas of the neocortex and the laminar distribution and the clustering of the tangles support the suggestion that the pathological changes in Alzheimer disease affect regions that are interconnected by well-defined groups of connections and that the disease process may extend along the connecting fibers. The invariable and severe involvement of the olfactory areas of the brain in this disease is in striking contrast to the minimal changes in the somatic sensory and primary visual areas and raises the possibility that the olfactory pathway may be initially involved.
Cloning and expression of apolipoprotein B, the major protein of low and very low density lipoproteins.
Authors: Lusis A J , West R , Mehrabian M , Reuben M A , LeBoeuf R C , Kaptein J S , Johnson D F , Schumaker V N , Yuhasz M P , Schotz M C
Published: May 2006

We report the cloning of cDNAs for rat liver apolipoprotein B (apo B) and the use of the cloned sequences to examine apo B expression at the level of mRNA in rat tissues. Fifteen putative apo B clones were identified by antibody screening of a rat liver cDNA library in the lambda gt11 expression vector. The identity of the clones was confirmed by immunological studies of the fusion protein products. All clones appear to contain sequences found only in apo B PI, the high molecular weight form of rat liver apo B. Blotting studies show that the clones hybridize to a single 20-kilobase liver mRNA species, sufficiently large to encode the entire apo B PI peptide, which is estimated to be 400 kDa in size. Apo B PI mRNA is abundant in liver and present in lower amounts in intestine but is absent in a variety of other tissues examined. This tissue distribution is consistent with that expected from studies on the in vivo synthesis of apo B. One clone, corresponding to a 240-base segment of the apo B PI mRNA, was sequenced and found to exhibit homology with a short region of rat apo E mRNA. Analysis of the secondary structure of the corresponding peptide did not show the preponderance of amphipathic alpha-helical structures characteristic of other apolipoproteins examined thus far.
Endothelial cells from bovine adrenal medulla develop capillary-like growth patterns in culture.
Authors: Banerjee D K , Ornberg R L , Youdim M B , Heldman E , Pollard H B
Published: May 2006

The endocrine barrier between chromaffin cells and the blood stream in the adrenal medulla is made of capillary endothelial cells. We have now succeeded in isolating endothelial cells from adrenal medullary tissue, which are probably derived from this barrier. These cells grow on plastic surfaces in the absence of special growth factors or collagen overlays and differentiate into organized structures quite similar to true capillaries. The cells contain factor VIII:R, a marker for endothelial cells, and form intercellular junctions characteristic of capillary endothelial cells. They also synthesize and secrete basal lamina structures and engage in transcytosis, a characteristic ultrastructural and functional combination of exocytosis and endocytosis across the thin endothelial cell processes. These endothelial cells can take up and deaminate catecholamines by A-type monoamine oxidase, an enzyme functionally distinct from the B-type monoamine oxidase found in chromaffin cells. These data indicate that the chromaffin cell and its endothelial cell neighbor may constitute the functional unit of catecholamine metabolism in the adrenal medulla.
Tetrahymena thermophila glutamine tRNA and its gene that corresponds to UAA termination codon.
Authors: Kuchino Y , Hanyu N , Tashiro F , Nishimura S
Published: May 2006

Nucleotide sequence analysis of one of several tRNA genes cloned from Tetrahymena thermophila macronuclear DNA indicated that it corresponds to a tRNA species having TTA as anticodon. Subsequently, the tRNA species corresponding to that gene was isolated and its nucleotide sequence was determined by post-labeling techniques. The nucleotide sequence was found to be pG-G-U-U-C-C-A-U-A-m2G-U-A-psi-A-G-D-G-G-D- D-A-G-U-A-C-U-G-G-G-G-A-Cm-U-Um-U-A-i6A-A-psi-C-C-C-U-U-G-A-C- m5C-U-G-G-G-U-psi-C-G-m1A-A-U-C-C-C-A-G-U-G-G-G-A-C-C-U-C-C-AOH. This tRNA sequence exactly matched the DNA sequence of the corresponding tRNA gene. The first position of anticodon is 2'-O-methyluridine (Um), forming UmUA as the anticodon, which presumably recognizes the ochre termination codon UAA. This tRNA species is aminoacylated with glutamine by a Tetrahymena crude aminoacyl tRNA synthetase fraction, suggesting that ochre termination codon is used as a glutamine codon during cytoplasmic protein synthesis in Tetrahymena.
Expression of a cloned denV gene of bacteriophage T4 in Escherichia coli.
Authors: Valerie K , Henderson E E , de Riel J K
Published: May 2006

A 713-base-pair Hae III fragment from bacteriophage T4 encompassing the denV gene with its preceding promoter has been cloned in a pBR322-derived positive-selection vector and introduced into a variety of DNA repair-deficient uvr and rec and uvr,rec Escherichia coli strains. The denV gene was found to be expressed, probably from its own promoter, causing pyrimidine dimer incision-deficient uvrA, uvrB, uvrC strains to be rescued by the denV gene. A uvrD (DNA helicase II) strain was also complemented, but to a lesser extent. A wild-type strain did not seem to be affected at the UV doses tested. Surprisingly, all recA, recB, and recC strains tested also showed an increased UV resistance, perhaps by reinforcement of the intact uvr system in these strains. Complementation of denV- T4 strains and host-cell reactivation of lambda phage was also observed in denV+ E. coli strains. Equilibrium sedimentation showed that DNA repair synthesis occurred in a UV-irradiated uvrA E. coli strain carrying the cloned denV gene. Southern blotting confirmed our earlier results [Valerie, K., Henderson, E. E. & de Riel, J. K. (1984) Nucleic Acids Res. 12, 8085-8096] that the denV gene is located at 64 kilobases on the T4 map. Phage T2 (denV-) did not hybridize to a denV-specific probe.
Mouse-Torpedo hybrid acetylcholine receptors: functional homology does not equal sequence homology.
Authors: White M M , Mayne K M , Lester H A , Davidson N
Published: May 2006

The nicotinic acetylcholine (AcCho) receptor (AcChoR) is a multisubunit protein complex of stoichiometry alpha 2 beta gamma delta. The several subunits show homology with each other within a given species; in addition, homology is found between analogous subunits between species. We have used the phage SP6 RNA polymerase transcription system to produce single-species RNA in vitro for various AcChoR subunits from cDNAs. Injection of an equimolar mixture of RNA for the alpha, beta, gamma, and delta subunits of Torpedo californica AcChoR into Xenopus oocytes results in the appearance of functional receptors in the oocyte membrane. No response to AcCho is detected when the beta or gamma subunit RNA is omitted, and a small response is seen when the delta subunit RNA is omitted. Replacement of Torpedo delta subunit RNA by the mouse BC3H-1 cell line AcChoR delta subunit RNA leads to the formation of functional receptors that show a 3-4-fold greater response to AcCho than does the full Torpedo complex. No response is seen when the mouse delta RNA replaces Torpedo gamma RNA. By amino acid homology profile comparisons, the mouse delta subunit appears to be moderately but not highly similar to the Torpedo delta subunit; the apparent similarity to the Torpedo gamma subunit is only slightly less. Therefore, the features of the primary sequence that determine the functional delta character of the mouse polypeptide are not revealed by simple homology comparisons.
Efficiency of utilization of various sources of energy for growth.
Authors: Donato K , Hegsted D M
Published: May 2006

The relative efficiency of dietary sucrose, protein sources, and fats in depositing body protein and fat (total energy) was directly estimated in young rats by feeding graded levels of each as supplements to a fixed amount of a basal diet that was presumably adequate in all essential nutrients except for energy. Under these conditions, the net gain in total body energy was a linear function of the amount of supplement added and the data fulfill the criteria of a valid slope-ratio bioassay. The available energy measured by this technique for sucrose and protein were similar, as would be expected. Dietary fat, however, was a more efficient source of energy. Compared to sucrose with 3.94 kcal/g (1 cal = 4.184 J), the average potency of dietary fat was 11.1 kcal/g, or approximately 124% of the expected value of 9 kcal/g. Fat supplements increased the deposition of body fat even when total energy intake was severely limited. The Atwater value of 9 kcal per g of fat is not appropriate under these conditions and probably not under other conditions.
A contact-insensitive subpopulation in Syrian hamster cell cultures with a greater susceptibility to chemically induced neoplastic transformation.
Authors: Nakano S , Ueo H , Bruce S A , Ts'o P O
Published: May 2006

We previously have identified a subpopulation of contact-insensitive (CS-) cells which lacks density-dependent inhibition of cell division in primary and low-passage cultures of Syrian hamster embryonic (SHE) fibroblastic cells. Further, we have shown that the proportion of these CS- cells declines as a result of the stable phenotypic conversion of the CS- cells to contact-sensitive (CS+) cells. To determine whether these transient CS- cells are more sensitive to carcinogenic/mutagenic perturbation, the susceptibility to neoplastic transformation and somatic mutation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined in clonally isolated cell cultures containing various proportions of CS- cells (0.02-4%). The frequencies of morphological transformation, focus formation, and neoplastic transformation showed a positive correlation to the proportion of CS- cells in the treated cultures. In contrast, the frequency of MNNG-induced somatic mutation at the Na+,K+-ATPase locus was similar among cultures varying in their proportion of CS- cells. Thus, there is a transient subpopulation of CS- cells in primary SHE cell cultures that is more susceptible to neoplastic transformation although equally susceptible to induced point mutation. This dissociation between somatic point mutation and neoplastic transformation indicates a fundamental difference in the nature of these two phenomena. A possible relationship between the propensity of CS- cells (versus CS+ cells) to carcinogen-induced neoplastic transformation and the state of differentiation of the CS- cells is discussed.
Antitermination by both the promoter and the leader regions of an Escherichia coli ribosomal RNA operon.
Authors: Holben W E , Prasad S M , Morgan E A
Published: May 2006

RNA polymerase initiating at Escherichia coli ribosomal RNA promoter-leader regions can efficiently read through factor rho-dependent termination signals. Dissection of the promoter-leader region reveals that the ability to read through termination signals is conferred independently by both promoter and leader regions. Events in the leader also affect the transcription rate of structural genes downstream of the leader. When cells are grown in rich medium, the rrnC leader reduces transcription by a factor of approximately 4 when downstream of the rrnC promoters and by a factor of 2 when downstream of the lac promoter.
Molecular cloning of Lyt-2, a membrane glycoprotein marking a subset of mouse T lymphocytes: molecular homology to its human counterpart, Leu-2/T8, and to immunoglobulin variable regions.
Authors: Nakauchi H , Nolan G P , Hsu C , Huang H S , Kavathas P , Herzenberg L A
Published: May 2006

The sequence of Lyt-2 cDNA shows that it is a new member of the immunoglobulin super gene family. Analysis of the predicted amino acid sequence indicates that the Lyt-2 polypeptide is synthesized with a 27-amino acid leader, and that the mature protein has an immunoglobulin variable region (Ig V)-related sequence of approximately 100 amino acids, an extracellular spacer of 43, a transmembrane region of 38, and an intracytoplasmic region of 27 amino acids. Lyt-2 and its human analogue Leu-2 are 56% homologous; analysis indicates that the Ig V-related domains of the two molecules have evolved away from each other faster than the carboxyl-terminal half of the proteins.
Disialoganglioside GD3 on human melanoma serves as a relevant target antigen for monoclonal antibody-mediated tumor cytolysis.
Authors: Cheresh D A , Honsik C J , Staffileno L K , Jung G , Reisfeld R A
Published: May 2006

Monoclonal antibody MB3.6 (IgG3) recognizes disialoganglioside GD3, which represents a major surface marker on most human melanoma cells. We demonstrate that this antibody effectively lyses four human melanoma cell lines expressing significant levels of GD3 on their surface by either of two mechanisms: antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated cytotoxicity. However, a melanoma cell line that expresses minimal levels of GD3 on 13% of the cells shows insignificant lysis by MB3.6 by either of these two mechanisms, suggesting that a threshold level of antigen expression may be required for effective in vitro cytolysis. In addition, monoclonal antibody (MAb) MB3.6 effectively inhibits establishment and growth of human melanoma tumors in the nude mouse when injected 24 hr after subcutaneous inoculation of tumor cells. Furthermore, MB3.6 produces specific regression of established melanoma tumors when injected 7 days after the subcutaneous inoculation of tumor cells. In contrast, tumor growth in animals injected with the melanoma cell line expressing minimal levels of GD3 was not affected by MAb MB3.6. These data indicate that once appropriate levels of the GD3 ganglioside are expressed on human melanoma cells, MAb MB3.6 can mediate tumor cell killing in vitro and in vivo and, thus, may prove useful for effective immunotherapy of human malignant melanoma.
Oxidant-dependent metabolic activation of polycyclic aromatic hydrocarbons by phorbol ester-stimulated human polymorphonuclear leukocytes: possible link between inflammation and cancer.
Authors: Trush M A , Seed J L , Kensler T W
Published: May 2006

Oxidants, such as those generated by metabolically activated phagocytes in inflammation, have been implicated in the metabolic activation of carcinogens, and in this study we demonstrate that the interaction of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP 7,8-dihydrodiol) with phorbol ester-stimulated polymorphonuclear leukocytes (PMNs) results in the generation of both a chemiluminescent intermediate and one that covalently binds to DNA. Cu(II)(3,5-diisopropylsalicylic acid)2 (CuDIPS), a biomimetic superoxide dismutase, and azide, a myeloperoxidase inhibitor, inhibited both of these reactions, indicating a dependency on oxygen-derived oxidants in these hydrocarbon-activation processes. Concordant with the formation of a carcinogen-DNA adduct, the admixture of BP 7,8-dihydrodiol and phorbol ester-stimulated PMNs elicited mutagenesis in Salmonella typhimurium strain TA100. 7,8-Dihydro-BP and BP cis-7,8-dihydrodiol were also mutagenic, whereas derivatives lacking a double bond at the 9,10 position were not. These results demonstrate that oxidants generated by metabolically stimulated PMNs can activate penultimate polycyclic aromatic hydrocarbons to a genotoxic metabolite and further defines a role for inflammation in carcinogenesis.
Brain and muscle nicotinic acetylcholine receptors are different but homologous proteins.
Authors: Conti-Tronconi B M , Dunn S M , Barnard E A , Dolly J O , Lai F A , Ray N , Raftery M A
Published: May 2006

An alpha-bungarotoxin-binding protein was purified from chick optic lobe and brain by an improved method. Previous and present observations justify its designation as a brain nicotinic acetylcholine receptor (AcChoR). It contains subunits whose apparent molecular weights are somewhat larger than those of subunits of peripheral AcChoRs. The size of the optic lobe AcChoR complex is greater than that of the peripheral receptor when estimated from its sedimentation behavior. Brain AcChoR subunits can be specifically precipitated by a monoclonal antibody directed against chick muscle AcChoR. Amino-terminal amino acid sequence analysis was performed on AcChoR preparations and isolated subunits from the optic lobe and from the rest of the chick brain. The sequences obtained demonstrate that, at least for the lowest molecular weight component, the AcChoRs from different brain areas are identical and they are highly homologous to muscle AcChoR. It is concluded that the brain alpha-bungarotoxin-binding protein is indeed a nicotinic AcChoR and is encoded by a set of genes that is different from, but strongly related to, that for the muscle AcChoR.
Cholinergic ventral forebrain grafts into the neocortex improve passive avoidance memory in a rat model of Alzheimer disease.
Authors: Fine A , Dunnett S B , Björklund A , Iversen S D
Published: May 2006

The memory dysfunction of Alzheimer disease has been associated with a cortical cholinergic deficiency and loss of cholinergic neurons of the nucleus basalis of Meynert. This cholinergic component of Alzheimer disease can be modeled in the rat by ibotenic acid lesions of the cholinergic nucleus basalis magnocellularis. The memory impairment caused by such unilateral lesions, as reflected in passive avoidance behavior, is reversed by grafts into the deafferented neocortex of embryonic neurons of the cholinergic ventral forebrain, but not by grafts of noncholinergic hippocampal cells.
High specific activity enantiomerically enriched juvenile hormones: synthesis and binding assay.
Authors: Prestwich G D , Wawrzeńczyk C
Published: May 2006

A stereoselective total synthesis of chiral juvenile hormone I is described that allows stoichiometric introduction of two tritium atoms in the final step. Both optical antipodes of the pivotal epoxy alcohol intermediate were prepared in 95% enantiomeric excess by the Sharpless epoxidation of a (Z)-allylic alcohol. Elaboration of the hydroxy-methyl group to a vinyl group followed by selective homogeneous tritiation affords optically active juvenile hormone I analogs at 58 Ci/mmol. Competitive binding of the labeled 10R, 11S and 10S,11R enantiomers with unlabeled enantiomers to the hemolymph binding protein of Manduca sexta larvae was determined by using a dextran-coated charcoal assay. The natural 10R,11S enantiomer has twice the relative binding affinity of the 10S,11R enantiomer. The availability of such high specific activity optically pure hormones will contribute substantially to the search for high-affinity receptors for juvenile hormones in the nuclei of cells. Moreover, the chiral 12-hydroxy-(10R,11S)-epoxy intermediate allows modification of juvenile hormone for solid-phase biochemical and radioimmunochemical work without altering either the biologically important carbomethoxy or epoxy recognition sites.
Vaccinia virus-infected cells release a novel polypeptide functionally related to transforming and epidermal growth factors.
Authors: Twardzik D R , Brown J P , Ranchalis J E , Todaro G J , Moss B
Published: May 2006

The recent discovery, that a vaccinia virus (VV) gene encodes a polypeptide with structural homology to transforming growth factor (TGF-alpha) and epidermal growth factor (EGF), led us to look for a virus-induced protein with the predicted biological activity. The supernatants of VV-infected cell cultures were found to contain an acid stable Mr 25,000 polypeptide that competes with EGF for binding to EGF membrane receptors. This VV-induced growth factor (VGF) like EGF and TGF-alpha is mitogenic and stimulates anchorage-independent cell growth in the presence of TGF-beta. However, VGF did not cross-react in a radioimmunoassay specific for small and large forms of TGF-alpha and exhibited minimal cross-reactivity with antisera to EGF. VGF was detectable in the culture medium within 2 hr, and maximal amounts were present 12 hr after infection. The level of VGF was proportional to the multiplicity of VV used. Inhibition of viral DNA synthesis enhanced VGF production, consistent with the hypothesis that VGF is an early gene product encoded by VV. The demonstration of a novel growth factor, released from cells infected with VV, may have important implications regarding the nature of virus-host interactions.
Accurate transcription of cloned Neurospora RNA polymerase II-dependent genes in vitro by homologous soluble extracts.
Authors: Tyler B M , Giles N H
Published: May 2006

We have developed soluble extracts from Neurospora crassa capable of accurately initiating the transcription of cloned Neurospora protein-encoding genes by RNA polymerase II in vitro. The genes encoding glutamate dehydrogenase (am) and histones H3 and H4 were transcribed by the extracts, and transcription was sensitive to alpha-amanitin at 1 mg/ml. The 5' heterogeneity of the in vitro initiation reactions was highly specific. Of the 17 transcription initiation sites within the inducible qa gene cluster, only one minor site was used in vitro, suggesting that, in general, transcription from qa gene promoters requires at least one different protein from those required for transcription of the am and histone genes.
Enhanced complement-mediated lysis of type III paroxysmal nocturnal hemoglobinuria erythrocytes involves increased C9 binding and polymerization.
Authors: Hu V W , Nicholson-Weller A
Published: May 2006

The interaction of terminal complement proteins (C5-C9) with normal erythrocytes and type III paroxysmal nocturnal hemoglobinuria erythrocytes (PNH-E) has been compared in terms of binding of the C5-9 complex, C9 polymerization, and C9 insertion into membranes. Complement components C5, C7, and C8 bind equally well to both types of erythrocytes, whereas the binding of C9 to PNH-E is 5-6 times greater than that to normal erythrocytes. The kinetics of C9 binding was compared with the kinetics of lysis for both types of cells under conditions leading to 100% lysis. There was a noticeable lag time between C9 binding and lysis of normal erythrocytes, but the lysis of PNH-E proceeded without a lag and the kinetics of lysis more closely paralleled C9 binding. The efficiency of C9 insertion was similar for both types of cells, but C9 polymerization was significantly enhanced on PNH-E. These data indicate that the enhanced susceptibility of type III PNH-E toward lysis by C5-9 can be correlated with abnormally high C9 binding and increased formation of poly(C9).
An unusual bovine pancreatic protein exhibiting pH-dependent globule-fibril transformation and unique amino acid sequence.
Authors: Gross J , Brauer A W , Bringhurst R F , Corbett C , Margolies M N
Published: May 2006

An unusual hitherto unreported protein, extracted in acid from fresh bovine pancreas, has been purified and characterized biochemically. It precipitates in the neutral pH range in the form of uniform double-helical threads, each strand of which is smooth and of uniform diameter, about 7-8 nm. The threads dissolve to a nonviscous solution below pH 3.6 and above pH 9.4, and they reconstitute reversibly in the pH range in between. The monomer in acid has an apparent molecular weight of 17,800 and consists of two disulfide-linked nonidentical polypeptide chains of different lengths. It is rich in aromatic amino acids, particularly tryptophan. There is no significant content of carbohydrate, fatty acid, or bound phosphate. The amino acid sequences of the first NH2-terminal 48 residues of the A chain and 35 residues of the B chain appear to be unique, differing from all other reported animal proteins, including those of the pancreas. Thus far, a function has not been found.
Molecular cloning of an activated human oncogene, homologous to v-raf, from primary stomach cancer.
Authors: Shimizu K , Nakatsu Y , Sekiguchi M , Hokamura K , Tanaka K , Terada M , Sugimura T
Published: May 2006

Transfection with high molecular weight DNA from a primary stomach cancer induced foci of transformed NIH 3T3 cells, and the transformed cells were tumorigenic in nude mice. By screening with a human Alu-family probe, we isolated the human DNA sequence from the secondary transformant cells. This transforming sequence encompasses about 60 kilobase pairs and is unrelated to known human transforming genes. Examination of homologies between this sequence and retroviral oncogenes revealed that the human transforming sequence is closely related to the v-raf oncogene of murine transforming retrovirus 3611-MSV.
Sterol control of the phosphatidylethanolamine-phosphatidylcholine conversion in the yeast mutant GL7.
Authors: Kawasaki S , Ramgopal M , Chin J , Bloch K
Published: May 2006

The relatively slow growth rate of the yeast mutant GL7, a sterol auxotroph, on medium containing cholesterol is markedly accelerated by supplementation with small amounts of ergosterol. Under these conditions (sterol synergism) cellular phospholipid synthesis is enhanced. We now find that one of the ergosterol-stimulated processes is the methylation of phosphatidylethanolamine to phosphatidylcholine. This is shown by comparing methyltransferase activities of membrane preparations derived from cells grown on either ergosterol, cholesterol, or the synergistic sterol pair. Incorporation of 32P from [gamma-32P]ATP into the yeast membranes is rapid and greater when ergosterol-grown cells rather than cholesterol-grown cells are the source of membranes.
Heparin-treated, v-myc-transformed chicken heart mesenchymal cells assume a normal morphology but are hypersensitive to epidermal growth factor (EGF) and brain fibroblast growth factor (bFGF); cells transformed by the v-Ha-ras oncogene are refractory to EGF and bFGF but are hypersensitive to insulin-like growth factors.
Authors: Balk S D , Riley T M , Gunther H S , Morisi A
Published: May 2006

Chicken heart mesenchymal cells do not proliferate in culture medium containing heat-defibrinogenated plasma but proliferate briskly when incubated with epidermal growth factor (EGF) or brain fibroblast growth factor (bFGF) plus insulin-like growth factors (IGFs) or when infected with sarcoma or erythroblastosis viruses. When infected with the retrovirus MC29, which bears a v-myc oncogene, chicken heart mesenchymal cells proliferate at a more modest rate and become morphologically transformed. Heparin at 25 microgram/ml causes these MC29-transformed cells to become proliferatively quiescent and to assume a normal morphology. Heparin-treated MC29-infected cells are, however, 100 times more sensitive to EGF than are their normal, uninfected counterparts. MC29-infected cells appear, likewise, to be hypersensitive to bFGF and to PDGF preparations but not to insulin. We hypothesize, therefore, (i) that heparin prevents the generation by cells of a mitogen from plasma protein precursors in the culture medium; (ii) that the v-myc oncogene renders cells hypersensitive to EGF, bFGF, PDGF, and the putative plasma-protein-derived mitogen; and (iii) that MC29-infected cells must proliferate in order to manifest the transformed morphology. Chicken heart mesenchymal cells infected with a recombinant spleen necrosis virus containing a v-ras oncogene are morphologically transformed but proliferate only sluggishly in plasma-containing medium without added mitogenic hormones. Heparin does not significantly affect their behavior. They are refractory to mitogenic stimulation by EGF or bFGF suggesting that ras proteins mediate the effects of receptors for these hormones. The SNV/v-ras-infected cells proliferate briskly, however, in response to hyperphysiological concentrations of insulin, an IGF surrogate, and are considerably more sensitive to this IGF mitogenicity than are their normal, uninfected counterparts.
In vitro screening assay for teratogens using growth inhibition of human embryonic cells.
Authors: Pratt R M , Willis W D
Published: May 2006

We have tested 35 teratogenic and 20 nonteratogenic chemicals or drugs in a short-term, in vitro assay that identifies teratogens by their ability to inhibit growth of an established line of human embryonic palatal mesenchymal cells. Only those chemicals that exhibited a dose-dependent inhibition of growth at concentrations less than 1 mM were classified as inhibitory. An Aroclor-induced rat liver S-9 system was effective in metabolizing cyclophosphamide to its teratogenic form in culture. We suggest that this assay, along with the complementary tumor cell-attachment assay of Braun et al. [Braun, A. G., Emerson, D. J. & Nichinson, B. B. (1979) Nature (London) 282, 507-509] may be useful as a short-term in vitro battery for assessment of the teratogenic potential in environmental agents and to prioritize those chemicals which merit further testing in vivo.
Intron sequences reveal evolutionary relationships among major histocompatibility complex class I genes.
Authors: Ronne H , Widmark E , Rask L , Peterson P A
Published: May 2006

The multigene family of the class I histocompatibility antigens is unusual in that allelic and intergenic differences often are of equal magnitude. It has been suggested that this is due to gene conversion events, which would produce allelic variation but at the same time reduce intergenic differences. We compared the sequences of 11 class I genes in an attempt to elucidate the evolutionary history of this gene family. Our analysis shows that the intron sequences can be used to establish the order of divergence of various class I genes from each other. The results obtained agree with the order of divergence deduced from major insertion and deletion events. It appears that certain genes in the murine TL antigen-encoding region diverged very early from the H-2 and Qa-2,3 genes. The latter can be subgrouped as H-2 and Qa-2,3 genes by both sequence homology and insertion patterns. In contrast to the introns, exon sequences provide less information on evolutionary relationships. Thus, these analyses are consistent with the view that concerted evolution due to gene conversion occurs preferentially in exons.
Differential effects of electrical stimulation of sciatic nerve on metabolic activity in spinal cord and dorsal root ganglion in the rat.
Authors: Kadekaro M , Crane A M , Sokoloff L
Published: May 2006

Electrical stimulation of the proximal stump of the transected sciatic nerve produces a frequency-dependent activation of glucose utilization, measured with the autoradiographic deoxy [14C]glucose method, in the dorsal horn of the spinal cord but produces no change in glucose utilization in the dorsal root ganglion cells. These results suggest that axon terminals and not the cell bodies are the sites of enhanced metabolic activity during increased functional activity of this pathway.
Role of the SulB (FtsZ) protein in division inhibition during the SOS response in Escherichia coli: FtsZ stabilizes the inhibitor SulA in maxicells.
Authors: Jones C , Holland I B
Published: May 2006

Induction of the SOS response in Escherichia coli by DNA-damaging treatments results in the synthesis of the SulA polypeptide, and this is sufficient to cause the resulting inhibition of cell division. Mutations at either sulA (sfiA) or sulB (sfiB) suppress this division inhibition. The SulB protein is identical to FtsZ, a protein required for normal division in E. coli. In the presence of FtsZ, the half-life of SulA synthesized in maxicells is approximately 12 min. In contrast, in the absence of FtsZ or in the presence of a mutant form of FtsZ (SulB114) that prevents division inhibition in vivo, SulA is extremely unstable with a half-life of only 3 min. Both FtsZ and SulA are isolated with the inner membrane of E. coli maxicells in the presence of MgCl2. We propose that the SulA inhibitor interacts directly with FtsZ in vivo to block the essential division function of this protein.
A possible mechanism of psoralen phototoxicity not involving direct interaction with DNA.
Authors: Laskin J D , Lee E , Yurkow E J , Laskin D L , Gallo M A
Published: May 2006

Psoralens in combination with ultraviolet light (UVA; 320-400 nm) are used in the photochemical treatment of a variety of skin diseases including vitiligo, a skin depigmentational disorder, and psoriasis, a disease of accelerated epidermal cell proliferation. Although it is generally assumed that the major site of action of the psoralens is DNA, we have obtained evidence that another site may be the primary target for these compounds. We have identified specific, saturable, high-affinity binding sites for 8-methoxypsoralen on HeLa cells and have detected specific binding of 8-methoxypsoralen to four other human cell lines and five mouse cell lines. In HeLa cells, specific binding is reversible and independent of the ability of the compound to intercalate into DNA. In addition, binding sites become covalently modified by the psoralen after UVA exposure. Specific binding of 8-[methoxy-3H]methoxypsoralen constitutes 79% of the label bound to the cells. Scatchard analysis indicated two classes of psoralen binding sites: high-affinity sites with a Kd of 19 X 10(-9) M (1.8 X 10(5) sites per cell) and low-affinity sites with a Kd of 4 X 10(-6) M (7.1 X 10(6) sites per cell). Four structurally related psoralen analogs block 8-methoxypsoralen binding in a manner that parallels their biological activity. Based on these findings, we hypothesize that specific binding sites for psoralens on mammalian cells mediate, at least in part, psoralen-induced phototoxicity.
Osteosarcoma and retinoblastoma: a shared chromosomal mechanism revealing recessive predisposition.
Authors: Hansen M F , Koufos A , Gallie B L , Phillips R A , Fodstad O , Brøgger A , Gedde-Dahl T , Cavenee W K
Published: May 2006

Survivors of the heritable form of retinoblastoma subsequently develop second primary osteosarcomas at substantially greater frequency than either the general population or survivors of nonheritable retinoblastoma. Here we present molecular genetic evidence that the development of these two disparate tumor types involves specific somatic loss of constitutional heterozygosity for the region of human chromosome 13 that includes the RB1 locus. Similar events occur during the genesis of nonheritable osteosarcoma but not in several other embryonal tumors or sarcomas. These findings suggest that a conceptual approach toward defining the number of genes whose recessive mutant forms predispose to cancer is the molecular genetic analysis of clinically associated tumor types. They also suggest that the molecular basis of mixed cancer families may be the differential expression of a single pleiotropic recessive mutation by tissue specific mitotic segregation abnormalities.
Dominant negative mutations in the Tn10 tet repressor: evidence for use of the conserved helix-turn-helix motif in DNA binding.
Authors: Isackson P J , Bertrand K P
Published: May 2006

The Tn10 tet repressor regulates transcription of the tetracycline-resistance determinant in transposon Tn10. Previous DNA sequencing studies identified a region of tet repressor (amino acids 26-47) that is homologous to the helix-turn-helix regions of lambda Cro, lambda repressor, and catabolite gene activator protein that are implicated in sequence-specific DNA binding. Here we report the isolation of dominant tetR mutations that result in tet repressors deficient in tet operator binding but that retain some capacity to form dimers with, and thereby inactivate, wild-type repressor monomers. The mutations were isolated by transforming a tetR+ tetA-lacZ fusion strain with hydroxylamine-mutagenized tetR plasmid DNA and then screening for increased lacZ expression. DNA sequence analysis of 35 independent isolates identified seven different mutations, five of which are in the region of helix-turn-helix sequence homology. In vitro binding studies indicate that the mutations in this region of tet repressor reduce the affinity of tet repressor for tet operator DNA by at least a factor of 1000 but have no significant effect on the affinity of tet repressor for tetracycline. These results provide strong support for the proposal that tet repressor utilizes the conserved helix-turn-helix structural motif in binding to tet operator DNA.
The specific induction of myc protooncogene expression in normal human B cells is not a sufficient event for acquisition of competence to proliferate.
Authors: Smeland E , Godal T , Ruud E , Beiske K , Funderud S , Clark E A , Pfeifer-Ohlsson S , Ohlsson R
Published: May 2006

Resting human B cells can be activated to proliferate in the presence of both polyclonal antibodies to immunoglobulin mu heavy chains and B-cell growth factor (BCGF). This process appears to be temporally controlled in that the initial activation of the B cells and their responsiveness to BCGF is carried out by polyclonal anti-mu-chain antibodies alone. We have used this system to investigate the role of the c-myc gene in the cell cycle of normal human peripheral blood B cells. Our results show that the polyclonal anti-mu-chain antibody-induced B-cell activation is accompanied by a specific induction of c-myc gene expression without promoting subsequent entry into the S phase unless BCGF is added. Monoclonal antibodies to either mu chain or the pan-B-cell antigen Bp35 also revealed a similar G0-to-G1 transition and activation of c-myc gene expression. However, unlike activation with polyclonal anti-mu-chain antibodies, cells stimulated with these monoclonal antibodies do not acquire responsiveness to BCGF. The results imply that additional inducible functions must be present to potentiate the myc-specific function in order for the B cells to acquire the capacity to proliferate in response to BCGF. These findings are discussed in relation to the origin of B-cell malignancies.
Dissociated cell culture of cholinergic neurons from nucleus basalis of Meynert and other basal forebrain nuclei.
Authors: Nakajima Y , Nakajima S , Obata K , Carlson C G , Yamaguchi K
Published: May 2006

Degeneration of cholinergic neurons from the basal forebrain nuclei is suspected to be the cause of Alzheimer disease. We have developed dissociated cultures of cholinergic neurons from these nuclei (the nucleus basalis of Meynert, the medial septal nucleus, and the diagonal band nuclei). Brain slices of the forebrains were made by a vibratome, and the basal forebrain nuclei were dissected out, dissociated, and cultured. Choline acetyltransferase immunocytochemistry and acetylcholinesterase cytochemistry revealed large cholinergic cells (average diameter, 20-25 micron) in these cultures. About 75% of large neurons (20 micron or larger in diameter) were cholinergic. Electrophysiological experiments were performed on these large neurons. The neurons usually did not show spontaneous firing, but steady depolarizations produced trains of action potentials, which adapted quickly. The neurons responded with depolarization to the application of L-glutamic acid. Substance P produced depolarization (sometimes hyperpolarization), and during the depolarization membrane resistance was increased.