bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro growth characteristics of simian T-lymphotropic virus type III.
Authors: Kannagi M , Yetz J M , Letvin N L
Published: May 2006

The type C retrovirus simian T-lymphotropic virus type III (STLV-III) has been isolated recently from immunodeficient macaque monkeys at the New England Regional Primate Research Center. The present studies were done to define the in vitro growth characteristics of this agent. STLV-III replicates efficiently in interleukin 2-dependent T-cell cultures of macaque peripheral blood lymphocytes (PBL), less efficiently in such cultures of human and gibbon PBL, and inefficiently in baboon PBL. No replication, as assessed by measuring reverse transcriptase activity in these culture supernatants, could be detected in similarly maintained cultures of chimpanzee, squirrel monkey, and cotton-top tamarin PBL. Like the human acquired immunodeficiency syndrome (AIDS) virus, human T-cell lymphotropic virus III/lymphadenopathy-associated virus (HTLV-III/LAV), STLV-III replicates in T4+ but not T8+ lymphocytes and its infection of macaque and human lymphocytes can be blocked with monoclonal anti-T4 antibodies. STLV-III differs from the human AIDS virus, however, in its apparent inability to grow in the Epstein-Barr virus-transformed B lymphocytes tested, the differing range of nonhuman primate T-cell populations that support its growth, and its less striking toxicity for T lymphocytes. These studies provide further characterization of an agent that will be extremely important in facilitating the development of vaccines and antiviral therapy for AIDS.
Membrane lipid changes in laminectomized and traumatized cat spinal cord.
Authors: Demediuk P , Saunders R D , Anderson D K , Means E D , Horrocks L A
Published: May 2006

Free fatty acid (FFA), diacylglycerol (acyl2Gro), icosanoid, phospholipid, and cholesterol levels were measured in samples of cat spinal cord (L2) that were frozen in situ with vertebrae intact, at various times after laminectomy, and at various times after laminectomy with compression trauma to the spinal cord. Tissue samples either were grossly dissected into gray and white portions prior to FFA and acyl2Gro analysis or were used whole for the other lipid types. Gray matter total FFA and acyl2Gro values were abnormally high in samples frozen with vertebrae intact and in those frozen 10 min after laminectomy. This indicates that the surgical procedures resulted in some perturbation of spinal cord lipid metabolism. If the experimental animals were allowed to recover for 90 min after laminectomy, the gray matter FFA and acyl2Gro levels were greatly reduced. Compression of the spinal cord with a 170-g weight for 1, 3, or 5 min (following 90 min of recovery after laminectomy) caused significant elevations of total FFA, acyl2Gro, icosanoids, and phosphatidic acid and significant decreases in ethanolamine plasmalogens and cholesterol. Among the total FFA, arachidonic acid was found to have the largest relative increase. Comparisons of gray and white matter demonstrate that, in general, changes in white matter FFA and acyl2Gro were similar to those seen in gray matter. However, the increases in white matter levels of FFA and acyl2Gro were delayed, occurring after the elevations in gray matter. For some FFA (e.g., arachidonate), the rise in white matter occurred as gray matter levels were decreasing. This suggests that the initial alteration in spinal cord lipid metabolism after trauma was in gray matter but, with time, spread radially into white matter.
Lithium perhydroantamanide complex (2:1): exposure of the peptide backbone in the analog of antamanide with antitoxic impotency.
Authors: Karle I L
Published: May 2006

The synthetic analog of antamanide in which all four phenylalanyl residues are hydrogenated to cyclohexylalanyl (Cha) residues, cyclic(Val-Pro-Pro-Ala-Cha-Cha-Pro-Pro-Cha-Cha), has a complete loss of antitoxic potency despite its ability to form ion complexes in the same manner as antamanide. The conformation of Li+.perhydroantamanide has been established in the present paper by x-ray diffraction analysis of a single crystal. The backbone encapsulates a Li+ ion in an almost identical manner as in Li+ antamanide. However, in Li+ antamanide the four phenyl groups are folded against the globular backbone, thus providing a hydrophobic surface for the complex, whereas in Li+ X perhydroantamanide the four cyclohexyl moieties are extended away from the folded backbone, resulting in the exposure of large portions of the polar backbone to the environment. As a consequence, four NH groups form hydrogen bonds with Br- ions, three C--O groups form hydrogen bonds with water molecules, and one C--O group makes a ligand to an additional external Li+ ion. The internal Li+ ion is pentacoordinated, whereas the external Li+ ion is tetracoordinated. The large change of the hydrophobicity around the midsection of the perhydroantamanide complex may be related to the biological inactivity. The content per asymmetric unit of the crystal is C64H102N10O10 X 2Li+ X 4H2O X 2CH3CN.2Br- in space group P2(1)2(1)2(1) with a = 21.740(7), b = 21.566(4), and c = 17.361(4) A. The agreement factor is 8.2% for 5135 data.
Crystal and molecular structure of chymotrypsin inhibitor 2 from barley seeds in complex with subtilisin Novo
Authors: McPhalen C. A. , Svendsen I. , Jonassen I. , James M. N. G.
Published: May 2006

The serine proteinase inhibitor from barley seeds, chymotrypsin inhibitor 2(CI-2), has been crystallized in a molecular complex with subtilisin Novo (EC 3.4.21.14). The crystal structure of this complex has been determined at 2.1-Å resolution by the molecular replacement method and partially refined by restrained-parameter least-squares methods. The present crystallographic
R
factor (Σ‖Fo[unk] - [unk]Fc‖/Σ[unk]Fo[unk]) is 0.193. CI-2 is a member of the potato inhibitor 1 family; it is a serine proteinase inhibitor lacking disulfide bonds. Comparison of the subtilisin molecule in this complex with the native subtilisin shows that the two molecules are very similar in structure. The inhibitor binds in a mode presumably resembling that of a true substrate, but it is not cleaved. This is in accord with the reported structures of other serine proteinase-inhibitor complexes. CI-2 consists of a four-stranded mixed parallel and antiparallel β-sheet against which an α-helix packs to form a hydrophobic core. A wide loop crossover connection between parallel strands 2 and 3 of the β-sheet contains the reactive-site bond. The conformation of the four residues to either side of the reactive-site bond is similar to that of the analogous residues in the third domain of the turkey ovomucoid inhibitor (Kazal family); the overall polypeptide chain fold of these inhibitors and the location of the reactive site in the respective chains are different.
A viral long terminal repeat in the interleukin 2 gene of a cell line that constitutively produces interleukin 2.
Authors: Chen S J , Holbrook N J , Mitchell K F , Vallone C A , Greengard J S , Crabtree G R , Lin Y
Published: May 2006

The gibbon leukemia cell line MLA 144 differs from every other T-lymphocyte line in that it constitutively makes interleukin 2 (IL-2) (also called T-cell growth factor) without stimulation by antigen, lectin, or tumor promoters. Previous work in which glucocorticoids were used to inhibit IL-2 production has indicated that proliferation of this cell line is dependent upon endogenously produced IL-2. We have found that the MLA 144 cell line has a copy of the gibbon leukemia virus inserted into the 3' nontranslated region of the IL-2 gene. This integration event produces a composite mRNA made up of the protein coding sequences of the IL-2 gene transcript but incorporating the viral long terminal repeat (LTR) in the 3' nontranslated region of the mRNA. This composite mRNA transcript uses the polyadenylylation signal in the viral 5' LTR and incorporates the viral transcriptional control regions. The integration event must involve only one allele of the IL-2 gene, since transcripts essentially identical to normal human IL-2 mRNA are also produced in cloned sublines of MLA 144. That the viral LTR contains a 94-base-pair repeat reminiscent of enhancer sequences in several viruses suggests that the integration of the viral LTR at the 3' end of the IL-2 gene is responsible for the constitutive production of IL-2 in the MLA 144 cell line.
Molecular cloning and nucleotide sequence of human glucocerebrosidase cDNA.
Authors: Sorge J , West C , Westwood B , Beutler E
Published: May 2006

Mutations in the human glucocerebrosidase gene cause Gaucher disease. A cDNA clone containing the entire human glucocerebrosidase coding region from normal cells has been isolated using lambda gt11 expression libraries. The complete nucleotide sequence, a restriction map, and a hydropathy profile are presented. Hybridization to chromosome-specific DNA localizes the human glucocerebrosidase gene to chromosome 1. The likely precursor protein is 515 amino acids long. The NH2-terminal 19 amino acids constitute a leader sequence that is cleaved from the mature protein. The predicted molecular weight of the mature protein is 55,384, without glycosylation or carboxyl-terminal processing.
Fabry disease: isolation of a cDNA clone encoding human alpha-galactosidase A.
Authors: Calhoun D H , Bishop D F , Bernstein H S , Quinn M , Hantzopoulos P , Desnick R J
Published: May 2006

Fabry disease is an X-linked inborn error of metabolism resulting from the deficient activity of the lysosomal hydrolase, alpha-galactosidase A (alpha-Gal A; alpha-D-galactoside galactohydrolase, EC 3.2.1.22). To investigate the structure, organization, and expression of alpha-Gal A, as well as the nature of mutations in Fabry disease, a clone encoding human alpha-Gal A was isolated from a lambda gt11 human liver cDNA expression library. To facilitate screening, an improved affinity purification procedure was used to obtain sufficient homogeneous enzyme for production of monospecific antibodies and for amino-terminal and peptide microsequencing. On the basis of an amino-terminal sequence of 24 residues, two sets of oligonucleotide mixtures were synthesized corresponding to adjacent, but not overlapping, amino acid sequences. In addition, an oligonucleotide mixture was synthesized based on a sequence derived from an alpha-Gal A internal tryptic peptide isolated by reversed-phase HPLC. Four positive clones were initially identified by antibody screening of 1.4 X 10(7) plaques. Of these, only one clone (designated lambda AG18) demonstrated both antibody binding specificity by competition studies using homogeneous enzyme and specific hybridization to synthetic oligonucleotide mixtures corresponding to amino-terminal and internal amino acid sequences. Nucleotide sequencing of the 5' end of the 1250-base-pair EcoRI insert of clone lambda AG18 revealed an exact correspondence between the predicted and known amino-terminal amino acid sequence. The insert of clone lambda AG18 appears to contain the full-length coding region of the processed, enzymatically active alpha-Gal A, as well as sequences coding for five amino acids of the amino-terminal propeptide, which is posttranslationally cleaved during enzyme maturation.
Serotonin neurons on the ventral brain surface.
Authors: Gorcs T J , Liposits Z , Palay S L , Chan-Palay V
Published: May 2006

Serotonin neurons and fibers on the subpial surface of the ventral medulla oblongata in the rat are described by immunohistochemistry and autoradiography. The neurons are concentrated in the area encompassed by the origins of the abducens, hypoglossal, glossopharyngeal, and vagus nerves. The highest number of serotonin surface neurons appears along the median medullary fissure or basilar sulcus, where they may represent the most ventral extensions of the raphe pallidus group. As these cells lie on the surface of the brain, they could be directly affected by alterations in the chemical composition of the cerebrospinal fluid and, depending on their connections, could influence important medullary functions.
Phosphorylation of the yeast equivalent of ribosomal protein S6 is not essential for growth.
Authors: Kruse C , Johnson S P , Warner J R
Published: May 2006

The yeast equivalent of ribosomal protein S6, known as S10, can be modified by the addition of two phosphates. The two adjacent serines that are likely to be subject to phosphorylation were deduced by comparison with the known sites of phosphorylation on rat liver S6. Using oligonucleotide mutagenesis, we altered the gene for S10 to replace these two serines with alanines. This mutant gene was introduced into a diploid yeast cell heterozygous for each of the two S10 genes. After sporulation, we obtained colonies in which the mutant gene was the only intact S10 gene. Although the ribosomes of these cells contained a full complement of S10, no phosphorylation of S10 was detected. These cells grow exponentially with a doubling time about 50% greater than that of control cells. We conclude that the phosphorylation of S10 is not essential for growth. However, the mutant gene in such cells is very unstable, frequently reverting to wild type, presumably by interaction with the disrupted host genes. We suggest that at some stage of the growth cycle there is strong selection for S10 that can be phosphorylated.
Incorporation of beef heart cytochrome c oxidase as a proton-motive force-generating mechanism in bacterial membrane vesicles.
Authors: Driessen A J , de Vrij W , Konings W N
Published: May 2006

Membrane vesicles derived from the strictly fermentative lactic acid bacterium Streptococcus cremoris have been fused with proteoliposomes containing the beef heart mitochondrial cytochrome c oxidase by means of a freeze/thaw-sonication technique. Evidence that fusion has taken place was obtained by freeze-etch electron microscopy, showing a less-dense intramembranous particle distribution in the fused membranes than in the bacterial membranes, and by sucrose gradient centrifugation, indicating a buoyant density of the majority of the membranes after fusion that was between the buoyant densities of the starting membrane preparations. In the fused membranes, 55-60% of the cytochrome c oxidase molecules are oriented with the cytochrome c binding site at the outer surface of the membrane. With the electron-donor system ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/cytochrome c, a high proton-motive force (greater than 130 mV), inside negative and alkaline, can be generated in the fused membrane, and this proton-motive force can drive secondary transport of several amino acids. The procedure described can be used for incorporating a proton-motive force-generating system in isolated membrane vesicles from bacterial or eukaryotic origin that lack a suitable primary proton pump.
Expected time for random genetic drift of a population between stable phenotypic states.
Authors: Lande R
Published: May 2006

Natural selection and random genetic drift are modeled by using diffusion equations for the mean phenotype of a quantitative (polygenic) character in a finite population with two available adaptive zones or ecological niches. When there is appreciable selection, the population is likely to spend a very long time drifting around the peak in its original adaptive zone. With the mean phenotype initially anywhere near the local optimum, the expected time until a shift between phenotypic adaptive peaks increases approximately exponentially with the effective population size. In comparison, the expected duration of intermediate forms in the actual transition between adaptive peaks is extremely short, generally below the level of resolution in the fossil record, and increases approximately logarithmically with the effective population size. The evolutionary dynamics of this model conform to the pattern of current paleontological concepts of morphological "stasis" and "punctuated equilibria."
Transition-state stabilization in the mechanism of tyrosyl-tRNA synthetase revealed by protein engineering.
Authors: Leatherbarrow R J , Fersht A R , Winter G
Published: May 2006

The principal catalytic factor in the activation of tyrosine by the tyrosyl-tRNA synthetase is found to be improved binding of ATP in the transition state. The activation reaction involves the attack of the tyrosyl carboxylate on the alpha-phosphate group of ATP to generate a pentacoordinate transition state. Model building of this complex located a binding site for the gamma-phosphate group of ATP, consisting of hydrogen bonds with the side chains of Thr-40 and His-45. Removal of these groups by protein engineering shows that they contribute no binding energy with unreacted ATP but put all of their binding energy into stabilizing the [tyrosine-ATP] transition state [the mutant tyrosyl-tRNA synthetase (Thr-40----Ala-40; His-45----Gly-45) has the rate of formation of tyrosyl adenylate lowered by 3.2 X 10(5) but KS for ATP is lowered by only a factor of 5]. The side chains of these residues also provide a binding site for pyrophosphate in the reverse reaction. Thus, catalysis is accomplished by stabilization of the transition state by improved binding of a group on the substrate that is distant from the seat of reaction.
Subunit stoichiometry and juxtaposition of the photosynthetic coupling factor 1: Immunoelectron microscopy using monoclonal antibodies
Authors: Tiedge Henri , Lünsdorf Heinrich , Schäfer Günter , Schairer Hans Ulrich
Published: May 2006

Monoclonal antibodies specific to the α subunits of the photosynthetic coupling factor 1 (CF
1
) were used as marker molecules in an electron microscopic analysis of the subunit organization of this enzyme. Immune complexes were obtained by incubation of CF
1
with saturating amounts of anti-α-subunit IgG, isolated by gel filtration, and visualized by electron microscopy. The maximum number of antibodies bound to a CF
1
molecule was three, the angle defined by a neighboring pair of antibodies characteristically being 120°. These results are interpreted as direct evidence for the presence of three α subunits in the CF
1
complex, the relative orientation of them being described by 3-fold rotary symmetry. Our observations thus favor an overall subunit stoichiometry of α
3
β
3
γδε.
Exposure of actin thiols by the removal of tightly held calcium ions.
Authors: Konno K , Morales M F
Published: May 2006

The removal of bound metal ions from G-actin uncovered two thiols, Cys-10 and Cys-257. The uncovering of these thiols requires a free calcium concentration lower than 10 nM. Therefore, participation of one or both thiols in Ca2+ binding is suggested. Actin labeled with N-(5-fluoresceinyl)maleimide in the absence of calcium moves as a doublet in NaDodSO4/PAGE. It is suggested that two conformers are induced by metal removal and labeling.
Complexes of polyoma virus medium T antigen and cellular proteins.
Authors: Grussenmeyer T , Scheidtmann K H , Hutchinson M A , Eckhart W , Walter G
Published: May 2006

Antibodies against synthetic peptides corresponding to the carboxyl-terminal six amino acids, Lys-Arg-Ser-Arg-His-Phe (KF), and an internal region, Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met-Glu (EE), of polyoma virus medium T antigen were used successively to purify medium T antigen by affinity chromatography. Medium T antigen from cell extracts was first bound to anti-KF antibodies and released from the immune complex with excess KF peptide; then it was bound to anti-EE antibodies and released with excess EE peptide. Two proteins, pp60c-src and a new protein of approximately equal to 61,000 Da (61-kDa protein), were copurified because they formed complexes with medium T antigen. The 61-kDa protein-medium T antigen complex was detected in extracts from wild-type-infected and transformed cells but not from cells infected with NG59 virus, which has a mutation in the medium T gene and is transformation defective. Instead, NG59 medium T antigen formed a complex with another cellular protein of approximately equal to 72,000 Da.
Disulfide bond formation in the eye lens.
Authors: Yu N T , DeNagel D C , Pruett P L , Kuck J F
Published: May 2006

The disposition and disposal of the -SH groups of the lens during aging and cataractogenesis have been investigated by laser Raman spectroscopy as a noninvasive microprobe in the intact living lens. In this procedure -SH and -S-S- give unique discrete Raman signals (at 2580 and 508 cm-1) that may be used to calculate relative concentrations in a very small volume of the lens. We present evidence showing an unexpected and remarkable difference with respect to these groups between the mouse lens and the lenses of guinea pig and man. The mouse lens nucleus exhibits a precipitous fall in the -SH concentration on aging from 1 to 6 months; concomitantly, there is a rise in -S-S- of comparable magnitude, indicating a direct conversion. The guinea pig lens, however, is quite different with respect to the age-dependent change in nuclear -S-S-: there is none between 6 months and 5 years. In the human lens -S-S- behaves exactly as in the guinea pig lens: the level is low and does not change with age between 9 and 65 years. With respect to nuclear -SH, these two latter species of lenses show some decrease with age but nothing like the approach to zero found in the aging mouse lens nucleus. These differences involving lenticular -SH and -S-S- appear to be correlated with the hard nucleus in the mouse lens and the softer nuclei of lenses in guinea pigs and humans. The relatively high level of -S-S- in the old but clear mouse lens does not support the idea that protein aggregation involving formation of intermolecular -S-S- bonds is necessarily an important cause of nuclear cataract. The small but significant age-related depression of -SH in guinea pig lens nuclei without any accumulation of -S-S- may be explained as a result of glutathione (GSH) oxidation and subsequent extrusion of glutathione disulfide (GSSG) by the lens. We propose that the oxidation of glutathione proceeds by reaction with protein disulfide groups to yield protein sulfhydryl (PSH) and a mixed disulfide of glutathione and protein; the mixed disulfide is capable of being reduced by glutathione reductase and NADPH, yielding the original PSH and GSSG, which is extruded from the lens. It remains to be determined if this mechanism is more active in guinea pig and human lenses than in the mouse lens.
Homologies between the Salmonella typhimurium CheY protein and proteins involved in the regulation of chemotaxis, membrane protein synthesis, and sporulation.
Authors: Stock A , Koshland D E , Stock J
Published: May 2006

Chemotactic receptors at the bacterial cell surface communicate with flagellar basal structures to elicit appropriate motor behavior in response to extracellular stimuli. Genetic and physiological studies indicate that the product of the cheY gene interacts directly with components of the flagellar motor to control swimming behavior. We have purified and characterized the Salmonella typhimurium CheY protein and have determined the nucleotide sequence of the cheY gene. Amino acid sequence comparisons showed CheY to be homologous over its entire length (129 residues) to the N-terminal regulatory domain of another protein involved in chemotaxis, the CheB methyl esterase. The entire CheY protein and the regulatory domain of CheB also homologous to the N-terminal portions of the Escherichia coli OmpR and Dye proteins and the Bacillus subtilis Spo0A protein. These homologies suggest an evolutionary and functional relationship between the chemotaxis system and systems that are thought to regulate gene expression in response to changing environmental conditions.
Isolation of cDNA clones encoding a T-cell receptor beta-chain from a beef insulin-specific hybridoma.
Authors: Morinaga T , Fotedar A , Singh B , Wegmann T G , Tamaoki T
Published: May 2006

cDNA clones coding for a T-cell receptor beta-chain were isolated from a beef insulin/IAd-reactive T-cell hybridoma, A20.2.15, and its complete amino acid sequence was deduced. This beta-chain gene utilizes the same V beta segment as a thymocyte beta-chain gene (86T1) and rearranges to the 5' proximal J-C locus (J beta 1-C beta 1), thus providing definitive evidence of a beta-chain gene from a functional hybridoma that utilizes C beta 1. The amino acid sequence of the V beta gene in A20.2.15 is identical to 86T1, thus suggesting the absence of somatic mutation in the beta-chain of A20.2.15. Southern blot analysis revealed a somatic DNA rearrangement unique to the A20.2.15 hybridoma. The expression of this gene in the hybridoma was confirmed by RNA dot hybridization. All 24 beta-chain clones so far isolated from the A20.2.15 hybridoma contained C beta 1, suggesting that the beta-chain gene of the fusion partner BW5147 is not expressed in the hybridoma.
Characterization of pituitary calcium-activated, phospholipid-dependent protein kinase: redistribution by gonadotropin-releasing hormone.
Authors: Naor Z , Zer J , Zakut H , Hermon J
Published: May 2006

We report the presence in the rat pituitary of a calcium-activated, phospholipid-dependent protein kinase (C kinase), originally described by Takai et al. [Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T. & Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695]. Enzyme activity is absolutely dependent on the simultaneous presence of Ca2+ and phospholipid--in particular, phosphatidylserine. The presence of small amounts of unsaturated diacylglycerol greatly increases the apparent affinity of the enzyme for Ca2+ and phosphatidylserine. Pituitary C kinase is mostly soluble (70%) and partly particulate (30%). Although the soluble form of the enzyme can be detected in a crude cytosol preparation, the particulate form is detectable only after solubilization and anion-exchange chromatography. Administration of a gonadotropin-releasing hormone (Gn-RH) agonist analog, [D-Ser(But)6]des-Gly10-Gn-RH-N-ethylamide, to ovariectomized rats resulted in elevated serum luteinizing hormone levels (245%) accompanied by a decrease in the cytosolic form of the enzyme (60%) and an increase in the particulate form (300%) after 5 min. This apparent activation of the particulate form seems to result from translocation of a soluble C kinase to the membrane. Several endogenous substrate proteins for C kinase ranging from 16 to 100 kDa were identified in pituitary cytosol. Pituitary C kinase might be involved in signal-transduction mechanisms in Gn-RH action, in particular, and in other hypophysiotropic hormones, in general, which operate by means of stimulation of phosphoinositide turnover during which diacylglycerol is liberated.
Corticotropin-releasing factor-induced adrenocorticotropin hormone release and synthesis is blocked by incorporation of the inhibitor of cyclic AMP-dependent protein kinase into anterior pituitary tumor cells by liposomes.
Authors: Reisine T , Rougon G , Barbet J , Affolter H U
Published: May 2006

Corticotropin-releasing factor (CRF) is the most potent and effective natural stimulant of corticotropin (ACTH) secretion. In a tumor cell line of the mouse anterior pituitary (AtT-20/D16-16) consisting of a homogeneous population of corticotrophs, CRF is known to increase adenylate cyclase and cAMP-dependent protein kinase activities as well as to release ACTH. To determine whether activation of cAMP-dependent protein kinase is essential for CRF to evoke the secretion of ACTH, an inhibitor (PKI) of this kinase was inserted into AtT-20 cells. This was accomplished by first encapsulating PKI into liposomes and then covalently coupling them to protein A for binding to antibodies directed against an AtT-20 cell surface antigen, N-CAM (neural cell adhesion molecule). The binding of the liposomes to the anti-N-CAM antibodies led to the internalization of the PKI into the tumor cells. The PKI treatment greatly attenuated CRF-stimulated ACTH release as well as the secretory response to beta-adrenergic agonists. However, ACTH release in response to caerulein, an agonist of cholecystokinin 8 receptors, was not altered by the PKI treatment. CRF treatment also increased the levels of mRNA for proopiomelanocortin (POMC), the precursor for ACTH in AtT-20 cells. Application of liposomes containing PKI to AtT-20 cells blocked the ability of CRF and 8-bromo-cAMP, but not phorbol ester, to increase POMC mRNA levels. The results revealed an essential role for cAMP in mediating the effect of CRF on ACTH release and POMC gene expression.
Modification of DNA by glucose 6-phosphate induces DNA rearrangements in an Escherichia coli plasmid.
Authors: Bucala R , Model P , Russel M , Cerami A
Published: May 2006

Reducing sugars such as glucose or glucose 6-phosphate (Glc-6-P) have been shown previously to modify the amino groups of nucleotides and single-stranded DNA. We have examined the mutagenic effect of Glc-6-P-induced lesions in the double-stranded DNA plasmid pBR322. Seventeen mutants of the Ampr Tets phenotype were isolated from plasmid preparations whose transforming capacity had been decreased by incubation with Glc-6-P. A number of the mutant plasmids were found to have undergone gross DNA alterations, including insertions and deletions, as well as the development of multiple species originating from a single cell. The ability of an endogenous reducing sugar to induce extensive DNA rearrangements suggests that these lesions may be significant contributors to cellular mutation.
Active site dynamics of ribonuclease.
Authors: Brünger A T , Brooks C L , Karplus M
Published: May 2006

The stochastic boundary molecular dynamics method is used to study the structure, dynamics, and energetics of the solvated active site of bovine pancreatic ribonuclease A. Simulations of the native enzyme and of the enzyme complexed with the dinucleotide substrate CpA and the transition-state analog uridine vanadate are compared. Structural features and dynamical couplings for ribonuclease residues found in the simulation are consistent with experimental data. Water molecules, most of which are not observed in crystallographic studies, are shown to play an important role in the active site. Hydrogen bonding of residues with water molecules in the free enzyme is found to mimic the substrate-enzyme interactions of residues involved in binding. Networks of water stabilize the cluster of positively charged active site residues. Correlated fluctuations between the uridine vanadate complex and the distant lysine residues are mediated through water and may indicate a possible role for these residues in stabilizing the transition state.
Localization of sialic acid in kidney glomeruli: regionalization in the podocyte plasma membrane and loss in experimental nephrosis.
Authors: Charest P M , Roth J
Published: May 2006

Sialic acid residues were localized by electron microscopy in renal glomeruli of normal and puromycin-treated rats with a cytochemical technique that utilized the Limax flavus lectin. In Lowicryl K4M thin sections from normal rats, sialic acid residues were found along the plasma membrane of the various glomerular cell types and in the glomerular basement membrane as well as the mesangial matrix. In NaDodSO4/PAGE, sialic acid residues of normal glomeruli were mainly confined to a 140-kDa protein previously identified as podocalyxin. The distribution of sialic acid residues in the podocyte plasma membrane was found to be remarkably regionalized. Based on the differential labeling intensity, three plasma membrane domains could be defined: the foot process base, the foot process region above the slit diaphragm, and the body of podocytes. Cytochemical and biochemical analysis of glomeruli from puromycin-treated rats showed a loss of sialic acid residues from glomerular sialoglycoconjugates indicating a perturbated glycosylation.
Human genes involved in cholesterol metabolism: chromosomal mapping of the loci for the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase with cDNA probes.
Authors: Lindgren V , Luskey K L , Russell D W , Francke U
Published: May 2006

Cellular cholesterol metabolism is regulated primarily through the coordinate expression of two proteins, the low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (EC 1.1.1.34). We have used cDNA probes for the human genes encoding these proteins to determine the precise chromosomal location of the two loci. By in situ hybridization we have regionally mapped the LDL receptor gene, LDLR, to the short arm of chromosome 19 in bands p13.1-p13.3. This result concurs with and extends a previous study in which LDLR was mapped to chromosome 19 by screening somatic cell hybrids with a species-specific monoclonal antibody. We have assigned the HMG-CoA reductase gene, HMGCR, to chromosome 5 by Southern blotting of DNA from a somatic cell hybrid panel and to bands 5q13.3-q14 by in situ hybridizations of the cDNA probe to human metaphase cells with normal and rearranged chromosomes.
Multiple-alphabet amino acid sequence comparisons of the immunoglobulin kappa-chain constant domain.
Authors: Karlin S , Ghandour G
Published: May 2006

We compare the amino acid sequences of the constant domains of the immunoglobulin kappa chain of human, mouse, and rabbit by using four classification schemes ("alphabets") of the 20 amino acids based on their chemical, functional, charge, and structural properties. The comparison reveals three regions of pronounced similarity across the three species, independent of allotype. Two of these regions (residues 65-73 and 99-103) entail a high degree of identity at the DNA level and are distinguished from the rest of the constant domain in codon usage and in the dinucleotide sequence at abutting sites of adjacent codons. Residues 22-29 are highly conserved among the three species in the chemical and functional alphabets but do not show any three-sequence significant amino acid block identities. These results are discussed in terms of transcript processing, effector functions, and structural interactions within the constant domain and with the heavy chain.
The lacI shuttle: rapid analysis of the mutagenic specificity of ultraviolet light in human cells.
Authors: Lebkowski J S , Clancy S , Miller J H , Calos M P
Published: May 2006

A system has been devised that allows the effect of mutagens acting in human cells to be readily analyzed at the DNA sequence level. The bacterial gene lacI, carried on a shuttle vector, is introduced into human tissue culture cells by transfection and allowed to replicate in the cell nucleus. Twenty-four to 48 hr after transfection, the cells are exposed to a mutagen. After 1-2 days of further replication, vector DNA is purified and transfected back into Escherichia coli for scoring and analysis of mutations in lacI. The nucleotide sequence changes for 53 UV light-induced mutations have been deduced in this way. Most of the mutations are transitions and occur at pyrimidine-pyrimidine sequences. The mutagenic specificity observed closely resembles that of UV light in E. coli, suggesting that human and bacterial cells respond similarly to damage from UV light. Use of the lacI shuttle in this way should permit determination of the mutagenic specificity of a wide range of mutagens and carcinogens in human cells.
Human T-cell growth factor (interleukin 2) and gamma-interferon genes: expression in human T-lymphotropic virus type III- and type I-infected cells.
Authors: Arya S K , Gallo R C
Published: May 2006

Acquired immune deficiency syndrome (AIDS) is characterized by severe depletion of OKT4+ T lymphocytes and leukemia is associated with abnormal proliferation of maturation-arrested lymphocytes. Human T-lymphotropic virus type III (HTLV-III) or lymphoadenopathy virus (LAV) and type I (HTLV-I) are etiologically linked to AIDS and adult T-cell leukemia/lymphoma, respectively. T-cell growth factor (TCGF, also known as interleukin 2) is required for the growth of activated T-cells, which play an important role in immune regulation. gamma-Interferon (IFN-gamma) is also implicated in immune modulation. It was possible that T-cell depletion in acquired immune deficiency syndrome could be due to an impairment of TCGF synthesis and that adult T-cell leukemia could be due to unregulated production of TCGF. The results reported here show that the transcription of the TCGF gene was not impaired in cultured HTLV-III-infected cells. Paradoxically, the TCGF gene in HTLV-I-infected cells was transcriptionally inactive. The reverse was the case for the gamma-interferon gene--it was actively transcribed in HTLV-I-infected cells but not in the HTLV-III-infected and virus-producing H9 and H4 cell line. No evidence was obtained suggesting abnormal regulation of the TCGF or of the IFN-gamma gene consequent to HTLV-III infection. It thus appears that in both HTLV-III and HTLV-I infection, growth control and immune regulatory mechanisms may bypass a modulatory role of TCGF or of IFN-gamma.
Human apolipoprotein E expression in Escherichia coli: structural and functional identity of the bacterially produced protein with plasma apolipoprotein E.
Authors: Vogel T , Weisgraber K H , Zeevi M I , Ben-Artzi H , Levanon A Z , Rall S C , Innerarity T L , Hui D Y , Taylor J M , Kanner D
Published: May 2006

Human apolipoprotein E (apoE) was produced in Escherichia coli by transforming cells with an expression vector containing a reconstructed apoE cDNA, a lambda PL promoter regulated by the thermolabile cI repressor, and a ribosomal binding site derived from the lambda cII or the E. coli beta-lactamase gene. Transformed cells induced at 42 degrees C for short periods of time (less than 20 min) produced apoE, which accumulated in the cells at levels of approximately equal to 1% of the total soluble cellular protein. Longer induction periods resulted in cell lysis and the proteolytic destruction of apoE. The bacterially produced apoE was purified by heparin-Sepharose affinity chromatography, Sephacryl S-300 gel filtration, and preparative Immobiline isoelectric focusing. The final yield was approximately equal to 20% of the initial apoE present in the cells. Except for an additional methionine at the amino terminus, the bacterially produced apoE was indistinguishable from authentic human plasma apoE as determined by NaDodSO4 and isoelectric focusing gel electrophoresis, amino acid composition of the total protein as well as its cyanogen bromide fragments, and partial amino acid sequence analysis (residues 1-17 and 109-164). Both the bacterially produced and authentic plasma apoE bound similarly to apolipoprotein B,E(low density lipoprotein) receptors of human fibroblasts and to hepatic apoE receptors. Intravenous injection resulted in similar rates of clearance for both the bacterially produced and authentic apoE from rabbit and rat plasma (approximately equal to 50% removed in 20 min). The ability to synthesize a bacterially produced human apolipoprotein with biological properties indistinguishable from those of the native protein will allow the production of large quantities of apoE for use in further investigations of the biological and physiological properties of this apolipoprotein.
The primary plasminogen-activator inhibitors in endothelial cells, platelets, serum, and plasma are immunologically related.
Authors: Erickson L A , Hekman C M , Loskutoff D J
Published: May 2006

Monospecific antiserum to an unusually stable Mr 50,000 plasminogen-activator inhibitor (PAI) purified from cultured bovine aortic endothelial cells was employed in conjunction with reverse fibrin autography to determine whether human platelets, serum, and plasma contain immunologically related inhibitors. Reverse fibrin autography revealed the presence of a Mr 50,000 inhibitor in the platelet and serum samples but not in normal plasma. However, a Mr 50,000 inhibitor was detected in plasma obtained from individuals with increased PAI activity. In each case, treatment of the sample with the anti-inhibitor serum removed the Mr 50,000 inhibitor. The inhibitor present in each sample neutralized exogenously added tissue-type plasminogen activator in a rapid manner. Inhibition was associated with the formation of a NaDodSO4-resistant enzyme-inhibitor complex of Mr 120,000. Again, treatment of the samples with the anti-inhibitor serum removed both the inhibitory activity and the component in these samples that binds to tissue-type plasminogen activator. Thus, the rapidly acting PAI present in human platelets, serum, and patient plasma is immunologically related to the PAI synthesized by cultured bovine aortic endothelial cells. This molecule may be the physiologically relevant inhibitor of plasminogen activator in the vascular system and, as such, may serve an important role in regulating the initiation of vascular fibrinolysis.
A genetic screen for mutations that increase the thermal stability of phage T4 lysozyme.
Authors: Alber T , Wozniak J A
Published: May 2006

A method has been developed to screen for mutants of phage T4 lysozyme that are more stable than the wild-type enzyme. Using an assay that detects lysozyme activity on Petri plates [Streisinger, G., Okada, Y., Emrich, J., Newton, J., Tsugita, A., Terzaghi, E. & Inouye, M. (1966) Cold Spring Harbor Symp. Quant. Biol. 31, 77-84], protein synthesized during the formation of phage plaques at a permissive temperature (33 degrees C) was tested for its ability to withstand incubation at a temperature that inactivates the wild-type enzyme. In our initial screen of approximately 3 X 10(4) plaques from a T4 phage stock mutagenized with hydroxylamine, greater than 30 mutants that produce lysozyme activity resistant to high temperature incubation were found. Lysozyme produced by two of the mutants was purified and found to denature at a higher temperature than the wild-type enzyme in vitro. We have called such mutants "st" for thermostable. The existence of st mutants indicates that protein stability is not maximized during evolution; instead, it is likely that stability is optimized for the physiology of the organism. Analysis of the structures of these mutants will provide another way to identify and predict interactions that stabilize proteins. The method of finding thermostable variants presented here may be applicable to any protein that can be detected by a plate assay or by a plate screen with antibodies.
Molecular dynamics simulations of d(C-G-C-G-A) X d(T-C-G-C-G) with and without "hydrated" counterions.
Authors: Singh U C , Weiner S J , Kollman P
Published: May 2006

We present the results of molecular dynamics simulations on d(C-G-C-G-A) X d(T-C-G-C-G) with fully charged phosphates with and without inclusion of counterions. The average structures found in the two simulations are similar, but the simulation with counterions does give an average helix repeat, tilt, and twist in better agreement with those found in the x-ray structure of d(C-G-C-G-A-A-T-T-C-G-C-G)2. The average sugar pucker phases and amplitudes are in qualitative agreement with those found in NMR studies of double-helical DNA, and a number of examples of sugar repuckering from C2' endo to C3' endo carbon conformations in the sugar ring are found. The hydrogen bond correlations as well as torsion correlations are analyzed, and some interesting long-range correlations between dihedral angles are found.
Determination of the stereostructure of the product of Tn3 resolvase by a general method.
Authors: Wasserman S A , Cozzarelli N R
Published: May 2006

A method has been developed for determination of the absolute structure of DNA catenanes. The catenated DNA is partially denatured before being thickened with a coating of RecA protein and spread for electron microscopy. This treatment allows visualization of the orientation of each ring as well as identification of the overlying and underlying DNAs at crossing points. These determinations define the topology of a catenane, providing a powerful means for testing mechanisms of catenane-producing enzymes in DNA recombination and replication. The technique was used to show that the single interlock of the catenated products of site-specific recombination mediated by Tn3 resolvase is exclusively of negative sign. The unique topology of the products indicates that resolvase fixes the sum of the number of supercoils between recombination sites at synapsis and the number of such supercoils lost or gained during strand exchange. The data strongly suggest that there are in fact three negative supercoils between synapsed sites; one supercoil is dissolved in the cross-over mechanism, whereas the other two are metamorphosed into the unique catenane interlock.
Somatic rearrangement of T-cell antigen receptor gene in human T-cell malignancies.
Authors: Minden M D , Toyonaga B , Ha K , Yanagi Y , Chin B , Gelfand E , Mak T
Published: May 2006

A cDNA clone representing the gene encoding the beta chain of the human T-cell antigen receptor has been isolated recently. By using fragments of this cDNA as hybridization probes in Southern blot analysis of restriction endonuclease-digested genomic DNA, we have now examined the structure of the gene in DNA from 26 patients with acute leukemia and from 23 normal individuals. We have found that the T-cell antigen receptor gene has undergone somatic rearrangement in 14 of 14 patients with the phenotypic diagnosis of T-cell acute lymphoblastic leukemia. In this group of patients, similar patterns of rearrangement appear to occur in different patients. This finding suggests that there is either a limited repertoire of possible rearrangements or an association between the development of leukemia and specific patterns of rearrangement. DNA from 6 patients with acute myeloblastic leukemia, 6 patients with non-B, non-T acute lymphoblastic leukemia, and 23 nonleukemic individuals showed no rearrangement or polymorphism. One case of T-cell acute lymphoblastic leukemia, however, showed rearrangement of both the T-cell receptor beta chain and the constant region of the immunoglobulin gene. Studies with mixtures of DNAs from leukemic bone marrow cells and cultured skin fibroblasts, as well as with remission and relapse marrow DNAs from the same patients, indicate that this technique can detect 1% leukemic cells in a mixed population. In addition, DNA from the marrow of a patient in relapse contains a similar rearrangement to that found in the marrow sample taken at the time of diagnosis, which suggests that the original clone of leukemic cells was responsible for relapse. Our results indicate that assessment of rearrangement of the T-cell antigen receptor gene will be valuable in the diagnosis and management of leukemia and can be used to evaluate clonality in T-cell neoplasia.
Involvement of the nerve growth factor-inducible large external glycoprotein (NILE) in neurite fasciculation in primary cultures of rat brain.
Authors: Stallcup W B , Beasley L
Published: May 2006

The nerve growth factor-inducible large external glycoprotein (NILE) has been found only on the surface of neuronal cells and Schwann cells. Since NILE seems to be concentrated on neurites, we have investigated its possible role in the development of neurites in primary cultures of rat brain. Cultures of embryonic day 14 (E14) whole brain and cultures of postnatal day 5 (P5) cerebellum were grown in the presence of Fab' fragments of antibody against NILE in an attempt to perturb the normal pattern of neurite development. For comparison, cultures were treated with two other reagents that recognize neuronal cell surface molecules: tetanus toxin, which binds to the GD1b and GT1 gangliosides, and Fab' fragments of antibody against neural cell adhesion molecule (N-CAM). Under the conditions used, none of the exogenous reagents affected neurite outgrowth, but specific effects on neurite fasciculation were observed. Anti-NILE inhibited fasciculation in cultures of E14 whole brain but had no effect on fasciculation in cultures of P5 cerebellum. Conversely, anti-N-CAM inhibited fasciculation in cultures of P5 cerebellum, which contain the adult form of N-CAM, but had little effect on fasciculation in cultures of E14 whole brain, which contain the embryonic form of N-CAM. Tetanus toxin had no effect on fasciculation in either culture system. Our results imply that NILE-mediated neurite-neurite interactions are stronger than N-CAM (embryonic)-mediated interactions in the E14 brain cultures, whereas N-CAM (adult)-mediated interactions are stronger than NILE-mediated interactions in the P5 cerebellar cultures.