bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of a reduced peptide inhibitor to the aspartic proteinase from Rhizopus chinensis: implications for a mechanism of action.
Authors: Suguna K , Padlan E A , Smith C W , Carlson W D , Davies D R
Published: May 2006

A peptide inhibitor, having the sequence D-His-Pro-Phe-His-Phe psi [CH2-NH]Phe-Val-Tyr, with a reduced bond between the two adjacent phenylalanines, has been diffused into crystals of the aspartic proteinase from Rhizopus chinensis (rhizopuspepsin, EC 3.4.23.6). X-ray diffraction data to 1.8-A resolution have been collected on the complex, which has been subjected to restrained least-squares refinement to an R-factor (R equals the sum of the absolute value of the difference between the observed and calculated structure factor amplitudes divided by the sum of the observed structure factor amplitudes) of 14.7%. The inhibitor lies within the major groove of the enzyme and is clearly defined with the exception of the amino-terminal D-histidine and the carboxyl-terminal tyrosine. The reduced peptide bond is located in the active site with close contacts to the two catalytic aspartyl groups. The active-site water molecule that is held between the two carboxyl groups is displaced by the inhibitor, as are a number of other water molecules seen in the binding groove of the native enzyme. A mechanism of action for this class of enzymes is proposed from these results.
Developmental, organ-specific, and light-dependent expression of the tomato ribulose-1,5-bisphosphate carboxylase small subunit gene family.
Authors: Sugita M , Gruissem W
Published: May 2006

The tomato gene family for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing); EC 4.1.1.39] has five genes, designated Rbcs-1, -2, -3A, -3B, and -3C. We have measured the steady-state mRNA levels for each of the five genes in various tomato organs using gene-specific oligonucleotides. All five genes are highly expressed in leaves, and transcripts of two genes, Rbcs-3B and Rbcs-3C, account for approximately equal to 60% of the total leaf transcripts. The relative transcript levels in the stem, in nature fruits, and etiolated seedlings (plants germinated and grown in the dark) correspond to 3.2%, 6.5%, and 4.6%, respectively, of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit mRNA level in leaves, and no transcripts have been detected in roots and ripe tomato fruits. Only Rbcs-1 and Rbcs-2 are expressed during the photosynthetically active phase of fruit development. Transcripts from these genes and from the Rbcs-3A locus are also present in etiolated seedlings. Rbcs-3B and Rbcs-3C transcripts, which are the most abundant mRNAs of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene family in the leaf, are undetectable in dark-grown seedlings and immature fruit. The selective expression of Rbcs-1 and Rbcs-2 in the dark and in the pericarp of green fruit and the induction and rapid mRNA accumulation for Rbcs-3B and Rbcs-3C after illumination may reflect different regulatory mechanism(s) that control the expression of individual members in the tomato ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene family.
Insertion and deletion mutagenesis of the human cytomegalovirus genome.
Authors: Spaete R R , Mocarski E S
Published: May 2006

Studies on human cytomegalovirus (CMV) have been limited by a paucity of molecular genetic techniques available for manipulating the viral genome. We have developed methods for site-specific insertion and deletion mutagenesis of CMV utilizing a modified Escherichia coli lacZ gene as a genetic marker. The lacZ gene was placed under the control of the major beta gene regulatory signals and inserted into the viral genome by homologous recombination, disrupting one of two copies of this beta gene within the L-component repeats of CMV DNA. We observed high-level expression of beta-galactosidase by the recombinant in a temporally authentic manner, with levels of this enzyme approaching 1% of total protein in infected cells. Thus, CMV is an efficient vector for high-level expression of foreign gene products in human cells. Using back selection of lacZ-deficient virus in the presence of the chromogenic substrate 5-bromo-4-chloro-3-indolyl beta-D-galactoside, we generated random endpoint deletion mutants. Analysis of these mutants revealed that CMV DNA sequences flanking the insert had been removed, thereby establishing this approach as a means of determining whether sequences flanking a lacZ insertion are dispensable for viral growth. In an initial test of the methods, we have shown that 7800 base pairs of one copy of L-component repeat sequences can be deleted without affecting viral growth in human fibroblasts.
Theoretical implications of cellular immune reactions against helper lymphocytes infected by an immune system retrovirus.
Authors: Reibnegger G , Fuchs D , Hausen A , Werner E R , Dierich M P , Wachter H
Published: May 2006

The breakdown of the immune system induced by the human immunodeficiency virus might be due to the active immune destruction of human immunodeficiency virus-infected helper T lymphocytes expressing viral antigens. By numerical simulation, we have studied possible consequences that a hypothetical immunodeficiency virus (IDV) may have on the cellular immune response by using a mathematical model. In this model, IDV infects CD4+ (helper) T cells and is actively synthesized by the immunologically activated helper T cells. Infected helper T cells synthesizing IDV express antigenic determinants specific for IDV and trigger a cellular immune response against themselves that is mediated by cytotoxic T cells and cytotoxic macrophages. The dynamic evolution of the model in the case of mixed-type infections with IDV and with another pathogen that evokes a cell-mediated immune response shows strong interactions between both simultaneous infections. The model might be of value to elucidate the dynamics leading to opportunistic infections. Furthermore, a pivotal role for immunological stimulation in the progressive exacerbation of the disease can be demonstrated.
Localization of neuropeptide Y mRNA in neurons of human cerebral cortex by means of in situ hybridization with a complementary RNA probe.
Authors: Terenghi G , Polak J M , Hamid Q , O'Brien E , Denny P , Legon S , Dixon J , Minth C D , Palay S L , Yasargil G
Published: May 2006

The distribution of mRNA encoding neuropeptide Y (NPY) in neurons of the normal human cerebral cortex in surgical biopsy specimens and postmortem brain was studied by in situ hybridization techniques. A 32P-labeled complementary RNA (cRNA) probe was used on cryostat sections of 13 formaldehyde-fixed cortical biopsy specimens. Hybridization to NPY mRNA was found in all samples: after autoradiography, discrete deposits of silver granules were observed on neuronal cell bodies abundantly distributed in the deep layers of the cortex, particularly laminae IV and VI, and on smaller cell bodies in the white matter. The localization of the neurons hybridized for NPY mRNA was comparable to that of NPY-immunoreactive cells as shown in sections from the same tissue blocks immunostained by using NPY antibodies. The specificity of the in situ hybridization technique was confirmed by blot hybridization analysis of electrophoretically fractionated RNA. This study clearly demonstrated the consistent localization of NPY gene transcription and expression in normal mature human cortical neurons.
Multiple DNA sequence elements are necessary for the function of an immunoglobulin heavy chain promoter.
Authors: Eaton S , Calame K
Published: May 2006

Sequences required for the function of the mouse V1 immunoglobulin heavy chain variable-region (VH) promoter were identified by transient transfection of the normal and mutated promoters into plasmacytoma cells. Our results identify four regions required for normal promoter function: (i) the octamer ATGCAAAT, previously identified by others; (ii) a heptamer, CTAATGA; (iii) a pyrimidine-rich region; and (iv) a region between positions -125 and -251 relative to the transcription start site. Sequence analysis of 19 mouse and human VH 5' flanking regions shows that the heptamer and pyrimidine stretch are strongly conserved. We have also demonstrated that the octamer functions in an orientation independent manner in the VH promoter.
Organization of the T-cell antigen-receptor beta-chain locus in mice.
Authors: Lindsten T , Lee N E , Davis M M
Published: May 2006

We used pulsed-field gel electrophoresis to determine the organization of the beta-chain gene of the T-cell receptor for antigen in normal and mutant inbred strains of mice. In normal mice, the variable (V)- and constant (C)- region elements of this locus span 700-800 kilobases of chromosomal DNA. All but one of the V beta gene segments analyzed lie 5' of the J beta C beta locus (J beta represents the joining region), with the closest being 280-360 kilobases away. The mutant mouse strain SJL has an internal V beta-region gene deletion that compacts the V beta region by 100-200 kilobases. Taken together with other data, these results indicate that the beta-chain locus can use either a looping-out/deletion or an inversion mechanism to appose V beta to DJ beta gene segments (D is the diversity region) and can accomplish the former (at least) over very large distances.
3H-labeled MK-801 binding to the excitatory amino acid receptor complex from rat brain is enhanced by glycine.
Authors: Reynolds I J , Murphy S N , Miller R J
Published: May 2006

We have studied the binding of the excitatory amino acid antagonist 3H-labeled MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine maleate] to extensively washed rat brain membranes. Binding of 3H-labeled MK-801 was inhibited by phencyclidine, Mg2+, and 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid in a manner dependent on the presence of L-glutamate or N-methyl-D-aspartate, suggesting that it labeled a site linked to the N-methyl-D-aspartate subtype of the glutamate receptor. Glycine also regulated 3H-labeled MK-801 binding, enhancing it in the concentration range of 0.01-10 microM. The actions of glutamate and glycine involved increases in binding affinity, without altering the number of 3H-labeled MK-801 binding sites. The effects of glycine in this system were mimicked by L- and D-alanine and L- and D-serine. However, beta-alanine and taurine were much less effective, and strychnine did not block the actions of glycine indicating that they were not mediated by the classical glycine receptors. Glycine also enhanced the ability of N-methyl-D-aspartate to increase Ca2+ influx into primary cultures of mouse striatal neurons measured using the Ca2+-sensitive fluorescent dye fura-2. These results support the suggestion that glycine may be an important regulator of the physiological actions of glutamate in vivo.
Cytosolic calcium transients from the beating mammalian heart.
Authors: Lee H C , Smith N , Mohabir R , Clusin W T
Published: May 2006

To elucidate the role of cytosolic calcium, [Ca2+]i, in the physiology of the normal and ischemic heart, we have developed a method for recording [Ca2+]i transients from the epicardial surface of the rabbit ventricle after arterial perfusion with the cell-permeant cytosolic calcium indicator indo-1 AM. Hearts were illuminated at 360 nm, and fluorescence was recorded simultaneously at 400 and 550 nm. The F400/F550 fluorescence ratio was calculated by an analog circuit that allowed cancelation of small movement artifacts that were present at single wavelengths. Clear [Ca2+]i transients were present in the F400/F550 signal and were remarkable for their slow decay. Slow decay of the transients was not due to buffering of [Ca2+]i by indo-1, since there was no associated impairment of contraction or relaxation. The peak amplitude of the [Ca2+]i transients was increased by ouabain, adrenaline, postextrasystolic potentiation, and acetylcholine. The extent to which the transients decayed diminished with shortening of the interbeat interval, but decay of the transients could be further diminished by acetylcholine or caffeine. A major advantage of the intact heart over isolated myocytes is the ability to measure changes in [Ca2+]i during ischemia. Ischemia produced a marked increase in both peak systolic and end-diastolic [Ca2+]i, which was most rapid during the first 30 sec, and approached a plateau value after 90 sec. This increase in [Ca2+]i was associated with a characteristic broadening of the peak of the transient. The increase in [Ca2+]i during ischemia is consistent with a proposed causative role of [Ca2+]i in mediating early electrophysiological abnormalities.
Human renin biosynthesis and secretion in normal and ischemic kidneys.
Authors: Pratt R E , Carleton J E , Richie J P , Heusser C , Dzau V J
Published: May 2006

The pathway of renin biosynthesis and secretion in normal and ischemic human kidneys has been investigated by pulse-labeling experiments. The results indicate that in normal human kidney, preprorenin is rapidly processed to 47-kDa prorenin. Microradiosequencing showed that this molecule was generated by cleavage between Gly-23 and Leu-24, yielding a 43-amino acid proregion. Analysis of prorenin secreted by the kidney tissue yielded an identical sequence, indicating that prorenin is secreted without any further proteolysis. An examination of the kinetics of processing and secretion suggested that a majority of the newly synthesized prorenin is quickly secreted, while only a small fraction is processed intracellularly to the mature renin. The differences in secretion kinetics between prorenin and mature renin and the selective inhibition of prorenin secretion by monensin suggest that they are secreted independently via two pathways: a constitutive pathway probably from the Golgi or protogranules that rapidly release prorenin and a regulated pathway that secretes mature renin from the mature granules. A comparison of the kinetics of processing between normal and ischemic tissues suggests that renal ischemia leads to an overall increase in the rate of processing of prorenin to mature renin. In addition, prolonged biosynthetic labeling of renin in the ischemic kidney yielded two smaller molecular weight immunoreactive forms suggestive of renin fragments that may be degradative products. These fragments were not detected in normal kidney tissue labeled for similar lengths of time.
Sources of propionate for the biogenesis of ethyl-branched insect juvenile hormones: Role of isoleucine and valine
Authors: Brindle Philip A. , Baker Fred C. , Tsai Leslie W. , Reuter Carol C. , Schooley David A.
Published: May 2006

Corpora allata from adult female
Manduca sexta
biosynthesize the sesquiterpenoid juvenile hormone (JH) III and the unusual ethyl-branched homologue JH II
in vitro
. We maintained corpora allata in medium 199 using [
methyl
-
3
H]methionine as the source of the JH methyl ester moiety and as a mass marker. This allowed measurement of the relative contributions of
14
C-labeled precursors to the biogenesis of JH II and III carbon skeletons. We showed efficient incorporation of a propionate equivalent, from isoleucine or valine catabolism, into the ethyl-branched portion of JH II, using double-label liquid scintillation counting of isolated JHs and gas chromatography/mass spectrometry with selected ion monitoring of JH deuteromethoxyhydrin derivatives. Methionine was a poor source of propionate for JH II biosynthesis, while glucose, succinate, threonine, and β-alanine did not contribute propionate at all. Leucine, isoleucine, and glucose incorporated into JH III and the acetate-derived portion of JH II.
Regulation of the induction of a cytochrome P-450 prostaglandin omega-hydroxylase by pregnancy in rabbit lung.
Authors: Muerhoff A S , Williams D E , Leithauser M T , Jackson V E , Waterman M R , Masters B S
Published: May 2006

The mechanism of induction of an adult rabbit cytochrome P-450, prostaglandin (PG) omega-hydroxylase (P-450PG-omega), during pregnancy has been investigated. This P-450 isozyme regiospecifically hydroxylates PGE1, PGA1, and PGF2 alpha at carbon-20 (the omega position). The specific activity of this enzyme is induced from 0.07 nmol of 20-OH-PGE1 to 3.05 nmol of 20-OH-PGE1 formed per min per mg of microsomal protein in the lungs of 25- to 28-day pregnant rabbits as compared to nonpregnant rabbits. Immunoblotting studies with a polyclonal antibody raised against this P-450 have shown that there is a concomitant gestational age-dependent increase in the amount of P-450PG-omega microsomal protein accompanying the increase of omega-hydroxylase activities. Within 3 days postpartum, both omega-hydroxylase activity and the amount of immunodetectable P-450PG-omega drop precipitously to near control levels. In vitro translation of total cellular RNA, extracted from the lungs of pregnant rabbits at various days throughout gestation, and immunoprecipitation of newly synthesized P-450PG-omega demonstrated a similar gestational age-dependent increase in translatable P-450PG-omega mRNA, as was observed with omega-hydroxylase activity and P-450PG-omega protein levels. These data suggest that the induction of this P-450 may occur at the transcriptional level and, furthermore, that control of cytochrome P-450PG-omega expression in the rabbit lung is tightly regulated at both protein and mRNA levels.
Heart tumors specifically induced in young avian embryos by the v-myc oncogene.
Authors: Saule S , Mérigaud J P , Al-Moustafa A E , Ferré F , Rong P M , Amouyel P , Quatannens B , Stéhelin D , Dieterlen-Lièvre F
Published: May 2006

To determine if expression of the v-myc oncogene had any effect during ontogeny, we injected avian myelocytomatosis virus strain MC29 into avian embryos at various stages of development. The injection of MC29 at embryonic day 2 (E2) or 3 (E3) caused, about 10 days later, rhabdomyosarcomas of the heart and, in some cases, skin muscle hypertrophy. When the injection was performed at E4 or E5, the number of heart tumors declined, whereas the number of skin muscle tumors increased significantly. The p110gag-myc protein was found in all tumors analyzed. When the virus was injected intravenously into E10 embryos, no tumors appeared during embryonic life, in striking contrast to the results obtained from injections at earlier stages. The monoclonal antibody 13F4, which is specific for the myogenic lineage, bound strongly to tumoral heart tissue, whereas it bound weakly to normal cardiac cells. Comparison of the peaks of tumor incidence in relationship to the timing of injection suggests that the v-myc product could interfere in vivo with an early step of the muscle lineage differentiation program. In addition, we show that the p58c-myc protein, which is supposed to play an important role in the control of cell proliferation, is only faintly detected in the heart of normal E3 embryos, in contrast to limb and tail buds, which readily express detectable levels of p58c-myc.
Molecular clock rates at loci under stabilizing selection.
Authors: Foley P
Published: May 2006

Under stabilizing selection in a finite population, lambda, the rate of allelic substitutions at a locus is approximately lambda approximately mu(1 + S)-1/2, where mu is the mutation rate. S, the stringency of selection upon new mutants, is defined by S = NVm/Vs, where N is the population size and Vm/Vs is a measure of the average fitness decrease experienced by new mutants. The approximation holds both for a "lonely" locus, which is the sole provider of genetic variation for the character under selection, and for an "embedded" locus, which is not. In both cases I use the Crow and Kimura model of a continuum of alleles with Gaussian selection and mutation. Monte Carlo simulations corroborate the substitution rate formula. Some molecular evolution data suggest the potential utility and limitations of the formula for estimating population size, mutation, and selection parameters. This work agrees with the rest of nearly neutral theory in emphasizing the important role of population size for substitution rates.
Interaction of tachykinins with their receptors studied with cyclic analogues of substance P and neurokinin B.
Authors: Ploux O , Lavielle S , Chassaing G , Julien S , Marquet A , d'Orléans-Juste P , Dion S , Regoli D , Beaujouan J C , Bergström L
Published: May 2006

The activities of two groups of cyclic agonists of substance P (SP) have been studied. The disulfide bridge constraints have been designed on the basis of conformational studies on SP and physalaemin indicating an alpha-helical structure for the core of these two tachykinins (group I) and a folding of the C-terminal carboxamide towards the side chains of the glutamines 5 and 6 (group II). Only peptides simulating the alpha-helix present substantial potencies. [Cys3,6]SP is as active as SP in inhibiting 125I-labeled Bolton and Hunter SP-specific binding on rat brain synaptosomes and on dog carotid bioassay, two assays specific for the neurokinin 1 receptor. Moreover, [Cys3,6]SP is as potent as neurokinin B in inhibiting 125I-labeled Bolton and Hunter eledoisin-specific binding on rat cortical synaptosomes as well as in stimulating rat portal vein, two tests specific for the neurokinin 3 receptor. Interestingly, in contrast to neurokinin B, [Cys3,6]SP is a weak agonist of the neurokinin 2 receptor subtype, as evidenced by its binding potency in inhibiting 3H-labeled neurokinin A-specific binding on rat duodenum and in inducing the contractions of the rabbit pulmonary artery, a neurokinin 2-type bioassay. To increase the specificity of the cyclic analogue [Cys3,6]SP positions 8 and 9 were modified. [Cys3,6, Tyr8, Ala9]SP is slightly less selective than SP for the neurokinin 1 receptor subtype. [Cys2,5]neurokinin B constitutes a selective cyclic agonist for the neurokinin 3 receptor. The very weak potencies of the peptides from group II indicate that a certain degree of flexibility in the C-terminal moiety is required. Collectively, these results suggest that the neurokinin 1 and neurokinin 3 tachykinin receptors may recognize a similar three-dimensional structure of the core of the tachykinins. Different orientations of the common C-terminal tripeptide may be related to the selectivity for the different receptor subtypes.
Prevention of occlusive coronary artery thrombosis by a murine monoclonal antibody to porcine von Willebrand factor.
Authors: Bellinger D A , Nichols T C , Read M S , Reddick R L , Lamb M A , Brinkhous K M , Evatt B L , Griggs T R
Published: May 2006

A murine monoclonal antibody (mAb) against porcine von Willebrand factor (vWF) induced an antithrombotic state in normal pigs. Thrombosis was induced by a standard procedure of stenosis and mechanical injury of the artery. The mAb was an IgG1 kappa that inhibited vWF-induced platelet aggregation at a titer of 1:6250 and bound to immobilized vWF at a maximal dilution of 1:512,000. The antibody did not affect two other vWF functions, platelet adhesion and binding of coagulant factor VIII (factor VIII:C). The antithrombotic state was characterized by a prolonged bleeding time and lack of plasma vWF activity, but with near-normal levels of factor VIII:C and von Willebrand antigen. The circulating Ag.mAb complex demonstrated a multimeric distribution comparable to that of native plasma vWF. Three groups of pigs were studied: group A consisted of nine untreated animals, eight of which developed occlusive coronary thrombosis; group B, four treated animals with a long bleeding time, none of which developed occlusive thrombosis; and group C, two animals with preexisting thrombosis treated with mAb, in which stable blood flow was reestablished. Morphologically, the group B animals showed adherent platelets covering the injured intima but no thrombosis. This mAb is an antithrombotic agent that prevents platelet thrombosis without affecting intrinsic platelet function.
Nucleotide sequence of testis-derived c-abl cDNAs: implications for testis-specific transcription and abl oncogene activation.
Authors: Oppi C , Shore S K , Reddy E P
Published: May 2006

The c-abl gene codes for a protein-tyrosine kinase and is expressed in most examined murine cell types as two distinct mRNA species of 5.5 kilobases (kb) and 6.5 kb. In mouse testis, an additional species of 4.0 kb is expressed in very high levels. To study the interrelationship between various c-abl transcripts and to compare their sequence with the v-abl transcript, we prepared c-abl-specific cDNA clones from mouse testis and determined the complete nucleotide sequence of the 4.0-kb cDNA that appears to be the reverse transcript of the testis-specific mRNA. In addition, we have determined the 3' sequence of an additional clone derived from the larger mRNA species that is expressed in somatic as well as germ-line cells. These cDNA sequences have been compared with the v-abl sequences to understand the mechanism of activation of this oncogene. The results demonstrate that (i) testis-specific c-abl mRNAs arise as a result of 3' truncation, and (ii) the v-abl gene has arisen from its cellular homologue as a result of an extensive deletional/mutational process.
Simian virus 40 large tumor antigen requires three core replication origin domains for DNA unwinding and replication in vitro.
Authors: Dean F B , Borowiec J A , Ishimi Y , Deb S , Tegtmeyer P , Hurwitz J
Published: May 2006

Simian virus 40 (SV40) large tumor antigen (T antigen) unwinds DNA containing the SV40 origin of replication. The origin requirement for unwinding can be satisfied by the 64-base-pair SV40 core origin that supports T-antigen-dependent DNA replication both in vivo and in vitro. The core origin contains three domains with specific DNA sequence features. These include an inverted repeat, a central T-antigen binding domain, and an adenine- and thymine-rich domain containing a DNA bending focus. The domain and spacer requirements of the core origin for DNA unwinding and replication in vitro are strikingly similar to the origin requirements for DNA replication in vivo. Thus, each of the three functional domains of the core origin contributes directly to the initiation of duplex DNA unwinding by T antigen.
Elevated glucose 6-phosphate levels are associated with plasmid mutations in vivo.
Authors: Lee A T , Cerami A
Published: May 2006

The incubation in vitro of plasmid pBR322 DNA with glucose 6-phosphate (Glc-6-P) has been shown to have a mutagenic effect when the plasmid was transformed into wild-type Escherichia coli. To further investigate the modifications of DNA by the reducing sugar Glc-6-P, we have developed an in vivo model to monitor plasmid DNA mutations. E. coli strains that are defective for phosphoglucose isomerase (strain DF40) alone or phosphoglucose isomerase and glucose-6-phosphate dehydrogenase (strain DF2000) accumulate Glc-6-P when grown in gluconate minimal medium in the presence of glucose. These strains and the control strain K10 were transformed with pAM006, a plasmid that carries the genes for ampicillin resistance and beta-galactosidase production, and grown for 24 hr under conditions that prompted the accumulation of Glc-6-P. An increase in plasmid mutations was observed (7- and 13-fold) that was associated with the increased intracellular levels of Glc-6-P (20- and 30-fold) present in the DF40 and DF2000 E. coli strains, respectively. Growth of the mutant bacteria in gluconate minimal medium does not increase the intracellular levels of Glc-6-P or the rate of plasmid mutations over background. Further characterization of the mutated plasmid DNA showed that insertions, deletions, and point mutations were responsible for the loss of beta-galactosidase production. The increase in plasmid mutations as a function of increased intracellular Glc-6-P levels suggests that the accumulation of adducts formed by Glc-6-P and other reducing sugars may contribute to DNA damage.
Conformational effects of substituting amino acids for glutamine-61 on the central transforming region of the P21 proteins.
Authors: Pincus M R , Brandt-Rauf P W , Carty R P , Lubowsky J , Avitable M , Gibson K D , Scheraga H A
Published: May 2006

The low-energy conformations for a series of peptides based on the sequence of the ras P21 protein from position 55 to position 67 have been computed using conformational energy analysis. These sequences differed at position 61 and contained Gln, Pro, Leu, Lys, and Arg at this position. P21 proteins with Gln, Glu, or Pro at this position do not cause cell transformation at normal levels of expression; proteins with substitutions of at least 14 other amino acids at this position (Leu, Lys, and Arg having been found in tumors in place of the normally occurring Gln-61) do cause malignant transformation of cells in culture. We find that the segments of residues 55-67 from the nontransforming proteins (Gln- or Pro-61) adopt a structure that is energetically unfavorable for the same segment with Leu, Lys, or Arg at position 61. The critical feature of this structure is an alpha-helix from residues 62 to 68. Residue 61 (Gln or Pro) adopts an extended conformation. On the other hand, the segment from transforming proteins can adopt two structures, one all alpha-helical from residue 61 to residue 68 and the other a less-regular, higher-energy structure. The segments from the normal protein can adopt the all alpha-helical structure, a finding that can explain the fact that elevated intracellular levels of the normal protein also cause cell transformation. The results of the calculations suggest that specific changes in the structure of this region can account for the oncogenic effect of the proteins in which substitutions occur.
Iron regulates ferritin mRNA translation through a segment of its 5' untranslated region.
Authors: Aziz N , Munro H N
Published: May 2006

In previous studies, we showed that acute administration of iron to intact rats or to rat hepatoma cells in culture induces synthesis of the iron-storage protein ferritin by activating translation of inactive cytoplasmic ferritin mRNAs for both the heavy (H) and the light (L) subunits. In the course of activation, these ferritin mRNAs are recruited onto polysomes. To elucidate the structural features of these mRNAs involved in the translational response to iron, a chimera was constructed from the 5' and 3' untranslated regions (UTRs) of ferritin L subunit mRNA fused to the reading frame of the mRNA of bacterial chloramphenicol acetyltransferase (CAT). This chimera and deletion constructs derived from it were introduced into a rat hepatoma cell line by retrovirus-mediated gene transfer. The complete chimera showed increased CAT activity in response to iron enrichment of the medium, whereas deletion of the first 67 nucleotides of the 5' UTR, which contain a highly conserved sequence, caused loss of regulation by iron. Whereas cis-acting sequences located in the 5' flanking regions of many genes have been repeatedly implicated in modulating their transcriptional expression, we report here a specific regulatory translational sequence found within the 5' UTR of a eukaryotic mRNA.
Deletional rearrangement in the human T-cell receptor alpha-chain locus.
Authors: de Villartay J P , Lewis D , Hockett R , Waldmann T A , Korsmeyer S J , Cohen D I
Published: May 2006

The antigen-specific receptor on the surface of mature T lymphocytes is a heterodimer consisting of polypeptides termed alpha and beta. In the course of characterizing human T-cell tumors with an immature (CD4-, CD8-) surface phenotype, we detected a 2-kilobase alpha-related transcript. Analysis of cDNA clones corresponding to this transcript established that a genetic element (which we call TEA, for "T early alpha") located between the alpha-chain variable- and joining-region genes had been spliced to the alpha constant region. The TEA transcript is present early in thymocyte ontogeny, and its expression declines during T-cell maturation. More important, the TEA area functions as an active site for rearrangement within the alpha gene locus. Blot hybridization of restriction enzyme-digested DNA with a TEA probe revealed a narrowly limited pattern of rearrangement in polyclonal thymic DNA, surprisingly different from the pattern expected for the mature alpha gene with its complex diversity. These DNA blots also showed that TEA is generally present in the germ-line configuration in cells expressing the gamma delta heterodimeric receptor and is deleted from mature (alpha beta-expressing) T-lymphocyte tumors and lines. Moreover, the TEA transcript lacked a long open reading frame for protein but instead possessed multiple copies of a repetitive element resembling those utilized in the heavy-chain class switch of the immunoglobulin genes. The temporal nature of the rearrangements and expression detected by TEA suggests that this recombination could mediate a transition between immature (gamma delta-expressing) T cells and mature (alpha beta-expressing) T cells.
Allogeneic H-2 antigen expression is insufficient for tumor rejection.
Authors: Cole G A , Cole G A , Clements V K , Garcia E P , Ostrand-Rosenberg S
Published: May 2006

Murine A strain (KkDdLd) sarcoma I (SaI) tumor cells have been transfected with a cloned H-2Kb gene. The resulting clones (SKB clones) stably express high levels of a molecule that is serologically and biochemically indistinguishable from the H-2Kb antigen. SKB clones are not susceptible to cytotoxic T lymphocyte-mediated lysis by H-2Kb-specific bulk, cloned, or H-2Kb-restricted lymphocytic choriomeningitis virus-specific effectors. Survival times of A/J and B10.A mice challenged i.p. with the H-2Kb-expressing transfectants and the parental SaI cells are similar, suggesting that the presence of an allogeneic major histocompatibility complex class I antigen on the surface of this tumor line is insufficient for tumor rejection.
Identification of a phenotype-specific enhancer in the first intron of the rat collagen II gene.
Authors: Horton W , Miyashita T , Kohno K , Hassell J R , Yamada Y
Published: May 2006

The regulation of the collagen II gene was investigated by transfecting plasmids containing potential regulatory sequences of this gene coupled to the gene for chloramphenicol acetyltransferase (CAT) into various cells. The 5' flanking region of this gene functioned as a weak promoter when transfected into chicken chondrocytes or fibroblasts. Inclusion of an 800-base fragment from the first intron, however, increased transcription of CAT approximately 18-fold in chicken chondrocytes and in differentiating limb bud cells but not in fibroblasts, myoblasts, muscle-derived fibroblasts, or teratocarcinoma cells. Furthermore, this fragment was active when placed either upstream or downstream of the CAT gene and when present in either orientation. The activity of this enhancer was examined in relation to differentiation by using "micromass" culture of limb bud mesenchyme cells undergoing chondrogenesis. In this system, no response to the enhancer was observed in undifferentiated limb bud mesenchyme cells. Following differentiation into chondrocytes, a 13-fold enhancer effect was observed in these cells. Finally, all-trans-retinoic acid, a known teratogen, dramatically suppressed enhancer activity in chondrocytes and differentiating limb bud mesenchyme cells. These results suggest that the collagen II gene contains an enhancer element in the first intron that is involved in cell-specific expression.
Differential expression of alternative 5' untranslated regions in mRNAs encoding rat insulin-like growth factor I.
Authors: Lowe W L , Roberts C T , Lasky S R , LeRoith D
Published: May 2006

Rat insulin-like growth factor I (IGF-I) cDNAs contain three alternative 5' untranslated sequences (termed class A, B, and C), which are associated with an identical coding region for the mature IGF-I peptide. A solution hybridization/RNase protection assay was used to simultaneously quantitate the relative abundance of IGF-I transcripts with the different 5' untranslated regions. In all the tissues studied, transcripts with the class C 5' untranslated region were most abundant. In contrast, both class A and B transcripts were tissue specific. Class A transcripts were present in moderate abundance in liver; in low abundance in kidney, lung, testes, and stomach; and were undetectable in muscle, heart, and brain; whereas class B transcripts were detected only in liver. These three classes of 5' untranslated region were also regulated independently by growth hormone. In liver, heart, kidney, and lung, growth hormone increased the abundance of class C transcripts 2- to 3-fold. In liver, growth hormone increased the abundance of the class A and B transcripts 6- to 7-fold. In lung and kidney, on the other hand, the abundance of class A transcripts was not affected by growth hormone. Thus, rat IGF-I gene transcripts contain one of three alternative 5' untranslated regions, which are expressed in a tissue-specific manner and are differentially regulated by growth hormone. Finally, cDNA probes unique to two of the three 5' untranslated regions hybridized to all three major species of IGF-I mRNA typically seen on RNA blots with a coding region probe.
Mobilization of T-DNA from Agrobacterium to plant cells involves a protein that binds single-stranded DNA.
Authors: Gietl C , Koukolíková-Nicola Z , Hohn B
Published: May 2006

Crude protein extracts of induced and uninduced octopine wild-type strain of Agrobacterium tumefaciens, as well as several mutants of the virulence loci virA, -B, -G, -C, -D, and -E, were probed with single- and double-stranded synthetic oligodeoxynucleotides of different sequence and length in an electrophoretic retardation assay. Four complexes involving sequence-nonspecific, single-stranded-DNA-binding proteins were recognized. One inducible complex is determined by the virE locus, two Ti-plasmid-dependent complexes are constitutively expressed, and a fourth one is controlled by chromosomal genes. The protein-DNA complexes were characterized by sucrose density gradient centrifugation and by determination of the length of single-stranded DNA required for their formation. It is hypothesized that the single-stranded-DNA-binding proteins are involved in the production of T-DNA intermediates or have a carrier or protective function during T-DNA transfer.
Unexpected binding of an octapeptide to the angiotensin II receptor.
Authors: Soffer R L , Bandyopadhyay S , Rosenberg E , Hoeprich P , Teitelbaum A , Brunck T , Colby C B , Gloff C
Published: May 2006

An octapeptide, TBI-22 (Lys-Gly-Val-Tyr-Ile-His-Ala-Leu), inhibited binding of angiotensin II by a solubilized angiotensin receptor partially purified from rabbit liver. This inhibition appears to result from competition for binding to the same receptor. Radioiodinated TBI-22, like angiotensin II, bound to the solubilized receptor with an affinity such that the binding was inhibited 50% by unlabeled TBI-22 or angiotensin II at nanomolar concentrations. The binding reaction, like that for angiotensin II, required p-chloromercuriphenylsulfonic acid and was reversed in the presence of dithiothreitol. TBI-22 and angiotensin II share the sequence Val-Tyr-Ile-His; this tetrapeptide alone, however, did not inhibit binding of angiotensin II. Replacement of the tyrosine residue by aspartic acid in TBI-22 greatly reduced the ability of the peptide to compete with angiotensin II for binding, suggesting an important contribution of this residue to the configuration required for recognition by the receptor.
Vitamin K-dependent carboxylase: possible role of the substrate "propeptide" as an intracellular recognition site.
Authors: Suttie J W , Hoskins J A , Engelke J , Hopfgartner A , Ehrlich H , Bang N U , Belagaje R M , Schoner B , Long G L
Published: May 2006

The liver microsomal vitamin K-dependent carboxylase catalyzes the posttranslational conversion of specific glutamate residues to gamma-carboxyglutamate residues in a limited number of proteins. A number of these proteins have been shown to contain a homologous basic amino acid-rich "propeptide" between the leader sequence and the amino terminus of the mature protein. Plasmids encoding protein C, a vitamin K-dependent protein, containing or lacking a propeptide region were constructed and the protein was expressed in Escherichia coli. The protein products were assayed as substrates in an in vitro vitamin K-dependent carboxylase system. Only proteins containing a propeptide region were substrates for the enzyme. These data support the hypothesis that this sequence of the primary gene product is an important recognition site for this processing enzyme.
Adenovirus type 2 endopeptidase: an unusual phosphoprotein enzyme matured by autocatalysis.
Authors: Chatterjee P K , Flint S J
Published: May 2006

A 19-kDa protein, present in low copy number in purified adenovirus type 2, has been characterized. Several criteria were used to establish that this protein is neither a degradation product of the known structural proteins of the virion nor a minor, unusually modified, form of protein VII. This 19-kDa protein, unlike other virion proteins, possesses alkali-resistant phosphoamino acids. Analysis by partial proteolysis indicated that it is related to a 23-kDa phosphoprotein present in H2ts-1 virions assembled in infected cells maintained at 39 degrees C. Affinity labeling with [3H]diisopropyl fluorophosphate showed that the 19-kDa protein contains the active site for a serine protease. We, therefore, conclude that the 19-kDa protein is the active form of the adenovirus-encoded endopeptidase, defined by the H2ts-1 mutation, and is synthesized as a 23-kDa precursor that appears to mature by autocatalysis.
Hormonal regulation of protein synthesis associated with salt tolerance in plant cells
Authors: Singh Narendra K. , LaRosa P. Christopher , Handa Avtar K. , Hasegawa Paul M. , Bressan Ray A.
Published: May 2006

Cultured tobacco cells (
Nicotiana tabacum
L. cv. Wisconsin 38) synthesize a predominant 26-kDa protein upon exposure to abscisic acid (ABA). ABA also accelerates the rate of adaptation of unadapted cells to NaCl stress. The ABA-induced 26-kDa protein is immunologically cross-reactive to, and produces a similar pattern of peptides after partial proteolysis as, the major 26-kDa protein associated with NaCl adaptation. Both have pI values of >8.2. The synthesis of the ABA-induced 26-kDa protein is transient unless the cells are simultaneously exposed to NaCl stress. There is an association between increased intracellular accumulation of ABA during cell growth and commencement of synthesis of the 26-kDa protein. ABA induces the synthesis of an immunologically cross-reactive 26-kDa protein in cultured cells of several plant species. In tobacco plants, synthesis of the 26-kDa protein could be detected in several tissues but the highest level of expression was seen in outer stem tissue. In root tissues, exogenous ABA greatly stimulated the synthesis of 26-kDa protein as compared to outer stem tissue and leaf. We suggest that ABA is involved in the normal induction of the synthesis of 26-kDa protein and that the presence of NaCl is necessary for the protein to accumulate.
Basic fibroblast growth factor in human rhabdomyosarcoma cells: implications for the proliferation and neovascularization of myoblast-derived tumors.
Authors: Schweigerer L , Neufeld G , Mergia A , Abraham J A , Fiddes J C , Gospodarowicz D
Published: May 2006

Cultured human embryonal rhabdomyosarcoma cells express the basic fibroblast growth factor (bFGF) gene and they produce bFGF, which is apparently composed of two microheterogenous forms with Mrs of 16,500 and 17,200, respectively. bFGF derived from the rhabdomyosarcoma cells stimulates their own proliferation and that of human or bovine vascular endothelial cells. It is conceivable that the rhabdomyosarcoma-derived bFGF stimulates the growth and neovascularization of human rhabdomyosarcomas and that it may thereby contribute to the development of these tumors.
Protective effects of analogs of luteinizing hormone-releasing hormone against x-radiation-induced testicular damage in rats.
Authors: Schally A V , Paz-Bouza J I , Schlosser J V , Karashima T , Debeljuk L , Gandle B , Sampson M
Published: May 2006

Possible protective effects of the agonist [D-Trp6]LH-RH (analog of luteinizing hormone-releasing hormone in which Gly-6 is replaced by D-tryptophan) and antagonist N-Ac-[D-Phe(pCI)1,2,D-Trp3,D-Arg6,D-Ala10]LH-RH against testicular damage caused by x-radiation were investigated in rats. Three months after being subjected to x-irradiation of the testes with 415 or 622 rads, control rats showed marked reduction in the weights of the testes and elevated levels of LH and follicle-stimulating hormone (FSH), indicating tubular damage. Histological studies demonstrated that, in testes of rats given 415 rads, most seminiferous tubules had only Sertoli cells and no germinal cells, and, in the group given 622 rads, the depression of spermatogenesis was even more marked. Rats pretreated for 50 days with LH-RH antagonist (1000 micrograms/kg of body weight per day) showed a complete recovery of testicular weights and spermatogenesis 3 months after 415 rads and showed partial recovery after 622 rads, and LH and FSH levels returned to normal in both of these groups. Thus, pretreatment of rats with LH-RH antagonist, by reversibly inhibiting gonadal function, protected the germinal cells of the testes against damaging effects of x-rays. Three experiments were also carried out in which the rats were pretreated for 1-2 months with long-acting microcapsules of the agonist [D-Trp6]LH-RH, liberating 25 micrograms of the agonist per day. Some rats were then subjected to gonadal irradiation with 415 or 622 rads and allowed a recovery period of 2-4 months. In spite of pretreatment with [D-Trp6]LH-RH, testicular weights were significantly lower and LH or FSH levels were elevated in the irradiated groups as compared with nonirradiated controls. The recovery of spermatogenesis was incomplete, and there was a decrease in the number of germinal cells after 415 rads and especially after 622 rads. On the basis of testicular weights, histology, and gonadotropin levels, it could be concluded that the agonist [D-Trp6]LH-RH did not protect the rat testes exposed to 622 rads and, at most, only partially protected against 415 rads. These results suggest that pretreatment with LH-RH antagonists and possibly agonists, might decrease the testicular damage caused by radiation and accelerate the recovery of reproductive functions.
A single base mutation in an I-A alpha-chain gene alters T-cell recognition.
Authors: Griffith I J , Choi E M , Glimcher L H
Published: May 2006

The interaction between the clonally selected T-cell antigen receptor, antigen, and Ia molecule is poorly understood at the molecular level. A cell line bearing an altered I-Ak alpha-chain (Ak alpha) molecule has been examined in order to provide more information about the relationship between Ia structure and function. The cell line, 3J9, was derived from the TA3 B-cell hybridoma through a series of negative and positive immunoselection steps. The 3J9 mutant lacked the binding site recognized by the Ak alpha-specific monoclonal antibody 39J and failed to present antigen to two T-cell hybridomas out of a large panel of I-Ak-restricted T-cell hybridomas examined. Sequence analysis of the mutant Ak alpha gene showed a single base transition (G----A) that resulted in a glutamic acid to lysine substitution at amino acid 75 of the alpha 1 domain. This mutation confirms the importance of amino acid 75 in the expression of the Ia.19 epitope, demonstrates the involvement of this region in the presentation of antigen to specific T cells, and provides a further example of the multiple functional domains on the Ia molecule that are involved in antigen presentation.