bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions.
Authors: Ylä-Herttuala S , Lipton B A , Rosenfeld M E , Särkioja T , Yoshimura T , Leonard E J , Witztum J L , Steinberg D
Published: May 2006

The recruitment of monocyte-macrophages into the artery wall is one of the earliest events in the pathogenesis of atherosclerosis. Monocyte chemoattractant protein 1 (MCP-1) is a potent monocyte chemoattractant secreted by many cells in vitro, including vascular smooth muscle and endothelial cells. To test whether it is expressed in the artery in vivo, we used Northern blot analysis, in situ hybridization, and immunocytochemistry to study the expression of MCP-1 in normal and atherosclerotic human and rabbit arteries. Northern blot analysis showed that MCP-1 mRNA could be isolated from rabbit atherosclerotic lesions but not from the intima media of normal animals. Furthermore, MCP-1 mRNA was extracted from macrophage-derived foam cells isolated from arterial lesions of ballooned cholesterol-fed rabbits, whereas alveolar macrophages isolated simultaneously from the same rabbits did not express MCP-1 mRNA. MCP-1 mRNA was detected by in situ hybridization in macrophage-rich regions of both human and rabbit atherosclerotic lesions. No MCP-1 mRNA was found in sublesional medial smooth muscle cells or in normal arteries. By using immunocytochemistry, MCP-1 protein was demonstrated in human lesions, again only in macrophage-rich regions. Immunostaining of the serial sections with an antiserum against malondialdehyde-modified low density lipoprotein indicated the presence of oxidized low density lipoprotein indicated the presence of oxidized low density lipoprotein and/or other oxidation-specific lipid-protein adducts in the same areas that contained macrophages and MCP-1. We conclude that (i) MCP-1 is strongly expressed in a small subset of cells in macrophage-rich regions of human and rabbit atherosclerotic lesions and (ii) MCP-1 may, therefore, play an important role in the ongoing recruitment of monocyte-macrophages into developing lesions in vivo.
Differential expression of two genes for 1-aminocyclopropane-1-carboxylate synthase in tomato fruits.
Authors: Olson D C , White J A , Edelman L , Harkins R N , Kende H
Published: May 2006

1-Aminocyclopropane-1-carboxylate synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14) is the regulated enzyme in the biosynthetic pathway of the plant hormone ethylene. A full-length cDNA encoding this enzyme has been cloned from tomato fruits [Van Der Straeten, D., Van Wiemeersch, L., Goodman, H. M. & Van Montagu, M. Proc. Natl. Acad. Sci. USA (1990) 87, 4859-4863]. We report here the complete nucleotide and derived amino acid sequences of a cDNA encoding a second isoform of ACC synthase from tomato fruits. The cDNAs coding for both isoforms contain highly conserved regions that are surrounded by regions of low homology, especially at the 5' and 3' ends. Gene-specific probes were constructed to examine the expression of transcripts encoding the two ACC synthase isoforms under two conditions of enhanced ethylene formation--namely, during fruit ripening and in response to mechanical stress (wounding). The level of mRNA encoding both isoforms, ACC synthase 1 and 2, increased during ripening. In contrast, wounding caused an increase in only the level of mRNA coding for ACC synthase 1. Blot analysis of genomic DNA digested with restriction enzymes confirmed that ACC synthase 1 and 2 are encoded by different genes.
Monoclinic uncomplexed double-stranded, antiparallel, left-handed beta 5.6-helix (increases decreases beta 5.6) structure of gramicidin A: alternate patterns of helical association and deformation.
Authors: Langs D A , Smith G D , Courseille C , Précigoux G , Hospital M
Published: May 2006

A comparison of the monoclinic and orthorhombic crystal structures of the uncomplexed double-stranded, antiparallel, left-handed beta-helix (5.6 amino acid residues per turn) (increases decreases beta 5.6) conformers of gramicidin A reveals marked differences in the tryptophan side-chain orientations and the degree of helical uniformity of the dimer and in the manner in which these helical dimers associate with one another in the crystal. The helix of the orthorhombic dimer exhibits a regular pattern of bulges and constrictions that appears to be induced by crystal packing forces affecting tryptophan side chains that are aligned parallel to the helix axis. The monoclinic dimer is more uniform than the orthorhombic dimer as a consequence of pi stacking interactions between dimers in which orientation of tryptophan side chains is normal to the helix axis to relieve the lateral crystal packing forces that may locally twist and deform the helix. It may be inferred from these observations that lipid interactions may be expected to destabilize the increases decreases beta 5.6 helix when it is inserted into a membrane bilayer.
Copper inhibits the protease from human immunodeficiency virus 1 by both cysteine-dependent and cysteine-independent mechanisms.
Authors: Karlström A R , Levine R L
Published: May 2006

The protease of the human immunodeficiency virus is essential for replication of the virus, and the enzyme is therefore an attractive target for antiviral action. We have found that the viral protease is inhibited by approximately stoichiometric concentrations of copper or mercury ions. Inactivation by Cu2+ was rapid and not reversed by subsequent exposure to EDTA or dithiothreitol. Direct inhibition by Cu2+ required the presence of cysteine residue(s) in the protease. Thus, a synthetic protease lacking cysteine residues was not inhibited by exposure to copper. However, addition of dithiothreitol as an exogenous thiol rendered even the synthetic protease susceptible to inactivation by copper. Oxygen was not required for inactivation of either the wild-type or the synthetic protease. These results provide the basis for the design of novel types of protease inhibitors.
Protonation of interacting residues in a protein by a Monte Carlo method: application to lysozyme and the photosynthetic reaction center of Rhodobacter sphaeroides.
Authors: Beroza P , Fredkin D R , Okamura M Y , Feher G
Published: May 2006

We used Monte Carlo methods to treat statistical problem of electrostatic interactions among many titrating amino acids and applied these methods to lysozyme and the photosynthetic reaction center of Rhodobacter sphaeroides, including all titrating sites. We computed the average protonation of residues as a function of pH from an equilibrium distribution of states generated by random sampling. Electrostatic energies were calculated from a finite difference solution to the linearized Poisson-Boltzmann equation using the coordinates from solved protein structures. For most calculations we used the Metropolis algorithm to sample protonation states; for strongly coupled sites, we substantially reduced sampling errors by using a modified algorithm that allows multiple site transitions. The Monte Carlo method agreed with calculations for a small test system, lysozyme, for which the complete partition function was calculated. We also calculated the pH dependence of the free energy change associated with electron transfer from the primary to the secondary quinone in the photosynthetic reaction center. The shape of the resulting curve agreed fairly well with experiment, but the proton uptake from which the free energy was calculated agreed only to within a factor of two with the observed values. We believe that this discrepancy resulted from errors in the individual electrostatic energy calculations rather than from errors in the Monte Carlo sampling.
Measurement of distinct immunochemical presentations of tau protein in Alzheimer disease.
Authors: Harrington C R , Mukaetova-Ladinska E B , Hills R , Edwards P C , Montejo de Garcini E , Novak M , Wischik C M
Published: May 2006

The tau protein is a microtubule-associated protein that is normally located in nerve axons. In Alzheimer disease, it is a constituent of paired helical filaments (PHFs), which are the principal fibrous component of the characteristic neurofibrillary tangles. The tau protein, therefore, is abnormally sequestered in an insoluble form in PHFs in the cell body and dendrites in Alzheimer disease. We have used two monoclonal antibodies (mAbs) to selectively measure the levels of normal, soluble tau protein and of PHF-associated tau protein in the brain. mAb 423 binds to PHFs and recognizes a 12-kDa fragment of tau protein released by formic acid treatment of PHFs, but it does not recognize normal tau protein. In contrast, mAb 7.51 recognizes normal tau protein as well as the PHF core-derived tau fragment, but its epitope is concealed in the PHF-bound form. The differential binding properties for these two mAbs have enabled us in this study to quantify insoluble PHF-associated tau protein in the somatodendritic compartment as well as normal soluble tau protein in its predominantly axonal location. Our findings demonstrate that a distinct immunochemical presentation of tau protein recognized by mAb 423, a PHF-specific marker, can be used to quantify neurofibrillary pathology in Alzheimer disease independently of the presence of normal tau proteins.
Adaptive evolution that requires multiple spontaneous mutations: mutations involving base substitutions.
Authors: Hall B G
Published: May 2006

A previous study has demonstrated that adaptive missense mutations occur in the trp operon of Escherichia coli. In this study it is shown that, under conditions of intense selection, a strain carrying missense mutations in both trpA and trpB reverts to Trp+ 10(8) times more frequently than would be expected if the two mutations were the result of independent events. Comparison of the single mutation rates with the double mutation rate and information obtained by sequencing DNA from double revertants show that neither our classical understanding of spontaneous mutation processes nor extant models for adaptive mutations can account for all of the observations. Despite a current lack of mechanistic understanding, it is clear that adaptive mutations can permit advantageous phenotypes that require multiple mutations to arise and that they appear enormously more frequently than would be expected.
Analysis of Krüppel control elements reveals that localized expression results from the interaction of multiple subelements.
Authors: Jacob Y , Sather S , Martin J R , Ollo R
Published: May 2006

The Drosophila gap gene Krüppel (Kr) displays a complex spatiotemporal pattern of expression during embryogenesis. Using P-element transformation experiments, we demonstrate that control elements guiding Kr expression in the central or in the anterior domain at the blastoderm stage are each composed of multiple subelements that interact synergistically. We provide evidence that bicoid (bcd) and hunch-back (hb) gene products, as well as at least one other activator, are needed to activate Kr expression in the central domain. We localize and describe regulatory elements within the 4.1-kilobase region proximal to the Kr promoter that are responsible for expression in the ectoderm, mesoderm, amnioserosa, and nervous system. Finally, a protein instability motif encoded in the second exon appears to be important for resetting the dynamic Kr pattern.
Enhanced insulin-like growth factor I gene expression in regenerating rat pancreas.
Authors: Smith F E , Rosen K M , Villa-Komaroff L , Weir G C , Bonner-Weir S
Published: May 2006

Insulin-like growth factor I (IGF-I) mRNA expression was studied after 90% partial pancreatectomy in the rat to determine whether IGF-I was associated with pancreatic regeneration. The level of IGF-I mRNA was maximally increased (4-fold above control value) 3 days after pancreatectomy, but thereafter gradually decreased, returning to control levels by 14 days after surgery. By in situ hybridization, IGF-I mRNA in both pancreatectomized and sham-operated rats was localized to capillary endothelial cells, indicating that this is the site of IGF-I expression in the normal rat pancreas. However, enhanced IGF-I mRNA expression was localized to focal areas of regeneration unique to pancreatectomized rats. In these areas, epithelial cells of proliferating ductules and individual connective tissue cells expressed IGF-I, suggesting that IGF-I may play an important role in the growth or differentiation of pancreatic tissue.
The subtypes of the mouse inositol 1,4,5-trisphosphate receptor are expressed in a tissue-specific and developmentally specific manner.
Authors: Nakagawa T , Okano H , Furuichi T , Aruga J , Mikoshiba K
Published: May 2006

Additional subtypes of the inositol 1,4,5-trisphosphate (InsP3) receptor are expressed in a tissue-specific and developmentally specific manner. They differ from the InsP3 receptor structure previously reported in two small variably spliced segments. One segment (SI) is located within the InsP3 binding site, whereas another segment (SII) is located near putative sites for phosphorylation and ATP binding to modulate InsP3 action on Ca2+ flux. Therefore, we speculate that selective use of InsP3 receptor subtypes permits a tissue-specific and developmentally specific expression of functionally distinct channels.
Molecular cloning and primary structure of Kell blood group protein.
Authors: Lee S , Zambas E D , Marsh W L , Redman C M
Published: May 2006

The Kell blood group is a major antigenic system in human erythrocytes. Kell antigens reside on a 93-kDa membrane glycoprotein that is surface-exposed and associated with the underlying cytoskeleton. We isolated tryptic peptides and, based on the amino acid sequence of one of the peptides and by using the PCR, prepared a specific oligonucleotide to screen a lambda gt10 human bone-marrow cDNA library. Four clones were isolated, one containing cDNA with an open reading frame for an 83-kDa protein. All known Kell amino acid sequences were present in the deduced sequence; moreover, rabbit antibody to a 30-amino acid peptide, prepared from this sequence, reacted on an immunoblot with authentic Kell protein. The Kell cDNA sequence predicts a 732-amino acid protein. Hydropathy analysis indicates a single membrane-spanning region, suggesting that Kell protein is oriented with 47 of its N-terminal amino acids in the cell cytoplasm, and a 665-amino acid segment, which contains six possible N-glycosylation sites, is located extracellularly. Computer-based search showed that Kell has structural and sequence homology to a family of zinc metalloglycoproteins with neutral endopeptidase activity.
Presence of activin (erythroid differentiation factor) in unfertilized eggs and blastulae of Xenopus laevis.
Authors: Asashima M , Nakano H , Uchiyama H , Sugino H , Nakamura T , Eto Y , Ejima D , Nishimatsu S , Ueno N , Kinoshita K
Published: May 2006

Activin A, a member of the transforming growth factor beta superfamily, has recently been found to have potent mesoderm-inducing activity on isolated early Xenopus animal-cap cells. We measured the activin activity of the Xenopus egg extract by using an erythroid-differentiating test with Friend leukemia cells. The results showed that an activin homologue is, indeed, contained in unfertilized eggs and blastulae of Xenopus laevis in a considerable amount. This activity was eluted at the same retention time as human activin A when fractionated by reversed-phase HPLC. Furthermore, the fraction containing erythroid-differentiating factor activity had mesoderm-inducing activity on Xenopus animal-cap cells. The mesoderm-inducing activity of this fraction was suppressed when coincubated with follistatin, an activin-binding protein. These results suggest that an endogenous activin may be a natural mesoderm-inducing factor acting in Xenopus embryogenesis.
Calcium ion as a cofactor in Na channel gating.
Authors: Armstrong C M , Cota G
Published: May 2006

Calcium ions in the external medium stabilize the resting state of voltage-dependent channels, including Na channels. This effect of calcium on channel gating is usually explained in terms of the surface charge hypothesis, which proposes that local adsorption of calcium ion to the outside of the membrane alters the intramembranous electric field, thus influencing channel behavior indirectly. Calcium ion has also been shown to block Na channels, most strongly at negative voltage. We have examined these two apparently separate effects of calcium, the gating effect and Ca block, and find the two are closely correlated. We propose that calcium (or a suitable substitute) is an essential cofactor in normal gating and that it produces gating and blocking effects by binding within the channel.
Ultra-small-angle x-ray-scattering study: preliminary experiments in colloidal suspensions.
Authors: Matsuoka H , Kakigami K , Ise N , Kobayashi Y , Machitani Y , Kikuchi T , Kato T
Published: May 2006

We report here a multiple-reflection type ultra-small-angle x-ray-scattering apparatus constructed according to the principle proposed by Bonse and Hart. The ultra-small-angle x-ray scattering technique enables us to investigate the distribution of solutes or particles in turbid systems, which could not be studied by the conventional light-scattering and microscopic methods. By this method, the density fluctuation from an order of several thousand angstroms to several micrometers is now detectable. In this paper, the ultra-small-angle x-ray scattering apparatus was calibrated by using well-defined latex particles.
Dietary fish oil blocks the microcirculatory manifestations of ischemia-reperfusion injury in striated muscle in hamsters.
Authors: Lehr H A , Hübner C , Nolte D , Kohlschütter A , Messmer K
Published: May 2006

Epidemiologic observations and experimental studies have demonstrated a protective effect of dietary fish oil on the clinical manifestations of ischemia-reperfusion injury. To investigate the underlying mechanisms, we used the dorsal skinfold chamber model for intravital fluorescence microscopy of the microcirculation in striated muscle of awake hamsters. In control hamsters (n = 7), reperfusion after a 4-hr pressure-induced ischemia to the muscle tissue elicited the adhesion of fluorescently stained leukocytes to the endothelium of postcapillary venules, capillary obstruction, and the break-down of endothelial integrity. These microvascular manifestations of ischemia-reperfusion injury were significantly attenuated in animals (n = 7) when fed with a fish oil-enriched diet for 4 weeks prior to the experiments. In leukocyte total lipids, the fish oil diet resulted in a substantial displacement of arachidonic acid, the precursor of the potent adhesion-promoting leukotriene (LT) B4, by fish oil-derived eicosapentaenoic acid, the precursor of biologically less potent LTB5, emphasizing the mediator role of LTB4 in ischemia-reperfusion injury. These results suggest that the preservation of microvascular perfusion by dietary fish oil contributes to its protective effects on the clinical manifestations of ischemia-reperfusion injury.
Histone contributions to the structure of DNA in the nucleosome.
Authors: Hayes J J , Clark D J , Wolffe A P
Published: May 2006

We describe the application of the hydroxyl radical footprinting technique to examine the contribution of the core histone tails and of histones H3 and H4 to the structure of DNA in the nucleosome. We first establish that, as was previously determined for a nucleosome containing a unique sequence of DNA, mixed-sequence nucleosomes contain two distinct regions of DNA structure. The central three turns of DNA in the nucleosome have a helical periodicity of approximately 10.7 base pairs per turn, while flanking regions have a periodicity of approximately 10.0 base pairs per turn. Removal of the histone tails does not change the hydroxyl radical cleavage pattern in either mixed- or unique-sequence nucleosome samples. A tetramer of histones H3 and H4, (H3/H4)2, organizes the central 120 base pairs of DNA identically to that found in the nucleosome. Moreover, "tailless" octamers and the (H3/H4)2 tetramer recognize the same nucleosome positioning signals as the intact octamer.
Interpulse interval in circulating growth hormone patterns regulates sexually dimorphic expression of hepatic cytochrome P450.
Authors: Waxman D J , Pampori N A , Ram P A , Agrawal A K , Shapiro B H
Published: May 2006

Plasma growth hormone (GH) profiles are sexually differentiated in many species and regulate the sex-dependence of peripubescent growth rates and liver function, including steroid hydroxylase cytochrome P450 expression, by mechanisms that are poorly understood. By use of an external pump to deliver to hypophysectomized rats pulses of rat GH of varying frequency and amplitude, a critical element for liver discrimination between male and female GH patterns was identified. Liver expression of the male-specific steroid 2 alpha (or 16 alpha)-hydroxylase P450, designated CYP2C11, was stimulated by GH at both physiological and nonphysiological pulse amplitudes, durations, and frequencies, provided that an interpulse interval of no detectable GH was maintained for at least 2.5 hr. This finding suggests that hepatocytes undergo an obligatory recovery period after stimulation by a GH pulse. This period may be required to reset a GH-activated intracellular signaling pathway or may relate to the short-term absence of GH receptors at the hepatocyte surface after a cycle of GH binding and receptor internalization. These requirements were distinguished from those necessary for the stimulation by GH of normal male growth rates in hypophysectomized rats, indicating that different GH responses and, perhaps, different GH-responsive tissues recognize distinct signaling elements in the sexually dimorphic patterns of circulating GH.
Production of interferon gamma, interleukin 2, interleukin 4, and interleukin 10 by CD4+ lymphocytes in vivo during healing and progressive murine leishmaniasis.
Authors: Heinzel F P , Sadick M D , Mutha S S , Locksley R M
Published: May 2006

The expression of interleukin (IL) 2, IL-4, IL-10, and interferon gamma (IFN-gamma) by lymphocyte subsets was examined during infection of resistant C57BL/6 and susceptible BALB/c mice with the protozoan parasite Leishmania major. CD4+ and CD8+ T lymphocytes and B lymphocytes were isolated from the lymph nodes draining infectious lesions, and their RNA was examined for lymphokine transcripts. Distinct patterns of CD4+ cell cytokine expression were apparent: C57BL/6 CD4+ cells contained IFN-gamma and IL-2 mRNA, whereas BALB/c CD4+ cells expressed IL-4 and IL-10 message. CD8+ cells contributed little lymphokine expression during disease, but B cells were a major source of IL-2 mRNA in both strains of mice. BALB/c mice made resistant by treatment with anti-CD4 antibody at the time of infection repopulated lymph nodes with CD4+ cells that expressed IL-2 and IFN-gamma. Protective treatment with anti-IL-4 antibody in vivo also resulted in the appearance of CD4+ cells with increased IFN-gamma and diminished IL-4 and IL-10 expression. These data establish CD4+ cells as the primary source of IFN-gamma in healing mice and of IL-4 and IL-10 during progressive infection and confirm that the spectral extremes of this disease are characterized by the presence of CD4+ cells expressing Th1 or Th2 phenotypes in vivo.
Protection of macaques with a simian immunodeficiency virus envelope peptide vaccine based on conserved human immunodeficiency virus type 1 sequences.
Authors: Shafferman A , Jahrling P B , Benveniste R E , Lewis M G , Phipps T J , Eden-McCutchan F , Sadoff J , Eddy G A , Burke D S
Published: May 2006

This report describes the vaccination of rhesus macaques with peptides selected from regions of the simian immunodeficiency virus (SIV) envelope that are hydrophilic, immunoreactive, and highly homologous with corresponding conserved envelope sequences of the human immunodeficiency virus (HIV). The peptides, produced as beta-galactosidase fusion proteins, induced virus-neutralizing and peptide-specific antibodies. After challenge with virulent virus, controls became virus positive and developed gradually rising antibody titers to SIV over 63 weeks. Immunized macaques developed a postchallenge anamnestic response to SIVenv antigens within 3-6 weeks followed by a gradual, fluctuating decline in SIV antibody titers and partial or total suppression of detectable SIV. Virus suppression correlated with prechallenge neutralizing antibody titers. Although the average CD4+ cell count in the blood of immunized macaques remained constant, the control macaques exhibited a progressive decrease developing about week 55 after challenge. The conserved nature of the HIV and SIV peptides and the similar humoral immunoreactivity in the respective hosts suggest that homologous HIV peptides may be important components of a successful immunization strategy.
Genetic analysis of microtubule motor proteins in Drosophila: a mutation at the ncd locus is a dominant enhancer of nod.
Authors: Knowles B A , Hawley R S
Published: May 2006

The nod (no distributive disjunction) and the ncd (non-claret disjunctional) mutations are both female-specific, recessive meiotic mutations in Drosophila melanogaster. Mutations at either locus show high frequencies of nondisjunction at meiosis I and both have been shown to encode kinesin-like proteins. Unlike the ncd mutation, which affects all chromosome pairs, the nod mutation affects only the disjunction of nonexchange chromosomes. Although both the nod and ncd mutations are fully recessive, females doubly heterozygous for nod and ncd mutations show levels of X and fourth chromosome nondisjunction that are 6- to 35-fold above those observed in control females. Exchange between chromosomes can suppress this effect; thus, only nonexchange chromosomes segregating via the distributive system are sensitive in double heterozygotes. Since the phenotype of double heterozygotes mimics that of the nod mutation, we infer that ncd is a dominant enhancer of nod. Failure of ncd to fully complement nod reveals the chromosome segregation machinery to be dosage sensitive. The probability that the distributive system will fail is enhanced in females simultaneously haploinsufficient at the nod and ncd loci.
A myelin protein is encoded by the homologue of a growth arrest-specific gene.
Authors: Welcher A A , Suter U , De Leon M , Snipes G J , Shooter E M
Published: May 2006

Striking features of the cellular response to sciatic nerve injury are the proliferation of Schwann cells in the distal nerve stump and the downregulation of myelin-specific gene expression. Once the axons regrow, the Schwann cells differentiate again to reform the myelin sheaths. We have isolated a rat cDNA, SR13, which is strongly downregulated in the initial phase after sciatic nerve injury. This cDNA encodes a glycoprotein that shares striking amino acid similarity with a purified myelin protein and is specifically precipitated by a myelin-specific antiserum. Immunohistochemistry experiments using peptide-specific polyclonal antibodies localize the SR13 protein to the myelin sheath of the sciatic nerve. Computer-aided sequence analysis identified a pronounced homology of SR13 to a growth arrest-specific mRNA (Gas-3) that is expressed in resting but not in proliferating 3T3 mouse fibroblasts. SR13 is similarly downregulated during Schwann cell proliferation in the rat sciatic nerve. The association of the SR13 as well as the Gas-3 mRNA with nonproliferating cells in two different experimental systems suggests a common role for these molecules in maintaining the quiescent cell state.
Mutant LexA proteins with an increased rate of in vivo cleavage.
Authors: Smith M H , Cavenagh M M , Little J W
Published: May 2006

LexA repressor of Escherichia coli is inactivated by a specific cleavage reaction that requires activated RecA protein in vivo. This cleavage reaction can proceed in vitro in the presence of activated RecA or as an intramolecular RecA-independent reaction, termed autodigestion, that is stimulated by alkaline pH. Here we describe a set of LexA mutant proteins that undergo a greatly increased rate of specific cleavage in vivo, compared with wild-type LexA. Efficient in vivo cleavage of these mutant proteins also took place without RecA. Several lines of evidence suggest that cleavage occurred via a mechanism similar to autodigestion. These mutations changed Gln-92, which lies near the cleavage site, to tyrosine, phenylalanine, or tryptophan. The latter mutation increased the rate of cleavage approximately 500-fold. These findings imply that the rate of wild-type LexA cleavage has been optimized during evolution to make the SOS system properly responsive to DNA-damaging treatments. Availability of these mutants will aid in the understanding of rate-limiting steps in intramolecular reactions.
Gene sequences suggest inactivation of alpha-1,3-galactosyltransferase in catarrhines after the divergence of apes from monkeys.
Authors: Galili U , Swanson K
Published: May 2006

The glycosylation enzyme alpha-1,3-galactosyltransferase (alpha 1,3GT; UDPgalactose:beta-D-galactosyl-1,4-N-acetyl-D-glucosaminide alpha-1,3-galactosyltransferase, EC 2.4.1.151) displays a unique pattern of distribution in mammals. It synthesizes an abundance of Gal(alpha 1-3)Gal(beta 1-4)GlcNAc-R (alpha-galactosyl) epitopes within the Golgi apparatus of cells of nonprimate mammals, prosimians, and New World monkeys (platyrrhines). The catarrhines, which include Old World monkeys, apes, and humans, lack this enzyme activity because of the inactivation of the alpha 1,3GT gene. In contrast, the catarrhines produce large amounts of antibodies, designated anti-Gal, against the alpha-galactosyl epitope. The inactivation of the alpha 1,3GT gene in ancestral catarrhines was probably the result of an intensive evolutionary pressure for alteration in the makeup of cell surface carbohydrates (i.e., suppression of alpha-galactosyl epitope expression) and for the production of the anti-Gal antibody. To determine the period in which the alpha 1,3GT gene was inactivated in ancestral catarrhines, comparative sequencing of a 370-base-pair region of this gene was performed by polymerase chain reactions with DNA of various primates. The data suggest that alpha 1,3GT inactivation occurred rather late in the course of catarrhine evolution (less than 28 million years ago), as separate events in apes and in Old World monkeys, after the two groups diverged from each other.
Parameters of the human genome.
Authors: Morton N E
Published: May 2006

Chromosome arm lengths are the critical parameters of the human genome. The physical length is required to scale radiation hybrid and other maps to megabases. The genetic lengths in males and females are required for probabilities of exclusion and synteny, choice of well-spaced loci for linkage tests, and comparison with centromeric maps based on nondisjunction. Interpolation of new data into a map is possible only when the length is known, including the distances from centromere and telomeres to the nearest markers. Current evidence on physical parameters including the reliable measurements of relative lengths from flow cytometry but only a crude estimate of genome size (3200 megabases). Evidence on genetic parameters includes chiasma counts and linkage maps corrected for failure to sample telomeres, giving an autosomal size of 2809 centimorgans in males and 4782 centimorgans in females. Estimates of the physical and sex-specific genetic lengths are presented for each chromosome arm. Any linkage analysis that yields substantially larger estimates raises a suspicion of an inappropriate mapping function or typing errors.
Human immunodeficiency virus induction of malignant transformation in human B lymphocytes.
Authors: Laurence J , Astrin S M
Published: May 2006

Aggressive B-cell lymphomas are occurring with increasing incidence among individuals infected with human immunodeficiency virus (HIV). Several lines of evidence implicate both Epstein-Barr virus (EBV) and c-myc activation in the pathogenesis of a major subset of these tumors. These observations prompted our investigation of interactions among EBV, c-myc, and HIV in primary B cells. We show that nonimmortalized peripheral B lymphocytes from EBV-seropositive, HIV-seronegative donors can be infected by HIV and that a subset of these lymphocytes become transformed. Malignant transformation was documented by several criteria. These cells displayed altered growth properties, propagating in 1% serum and cloning in soft agar, and formed invasive tumors of Burkitt lymphoma phenotype after subcutaneous injection into severe combined immunodeficiency mice. Such cells revealed marked enhancement of EBV DNA and RNA and of endogenous c-myc transcripts and protein. HIV-1 infection of already immortalized B-cell lines led to a similar upregulation of EBV and c-myc transcripts. These data indicate that HIV has properties of a transforming retrovirus, as it mediates two events linked to B-cell neoplasia: deregulation of c-myc and activation of EBV. They also raise the possibility of a role for HIV, apart from induction of immune suppression, in the pathogenesis of B-cell lymphoma in the acquired immune deficiency syndrome.
Expression of a partially deleted gene of human type II procollagen (COL2A1) in transgenic mice produces a chondrodysplasia.
Authors: Vandenberg P , Khillan J S , Prockop D J , Helminen H , Kontusaari S , Ala-Kokko L
Published: May 2006

A minigene version of the human gene for type II procollagen (COL2A1) was prepared that lacked a large central region containing 12 of the 52 exons and therefore 291 of the 1523 codons of the gene. The construct was modeled after sporadic in-frame deletions of collagen genes that cause synthesis of shortened pro alpha chains that associate with normal pro alpha chains and thereby cause degradation of the shortened and normal pro alpha chains through a process called procollagen suicide. The gene construct was used to prepare five lines of transgenic mice expressing the minigene. A large proportion of the mice expressing the minigene developed a phenotype of a chondrodysplasia with dwarfism, short and thick limbs, a short snout, a cranial bulge, a cleft palate, and delayed mineralization of bone. A number of mice died shortly after birth. Microscopic examination of cartilage revealed decreased density and organization of collagen fibrils. In cultured chondrocytes from the transgenic mice, the minigene was expressed as shortened pro alpha 1(II) chains that were disulfide-linked to normal mouse pro alpha 1(II) chains. Therefore, the phenotype is probably explained by depletion of the endogenous mouse type II procollagen through the phenomenon of procollagen suicide.
A prion-like protein from chicken brain copurifies with an acetylcholine receptor-inducing activity.
Authors: Harris D A , Falls D L , Johnson F A , Fischbach G D
Published: May 2006

The mammalian prion protein (PrPC) is a cellular protein of unknown function, an altered isoform of which (PrPSc) is a component of the infectious particle (prion) thought to be responsible for spongiform encephalopathies in humans and animals. We report here the isolation of a cDNA that encodes a chicken protein that is homologous to PrPC. This chicken prion-like protein (ch-PrLP) is identical to the mouse PrP at 33% of its amino acid positions, including an uninterrupted stretch of 24 identical residues, and it displays the same structural domains. In addition, ch-PrLP, like its mammalian counterpart, is attached to the cell surface by a glycosyl-phosphatidylinositol anchor. We find that ch-PrLP is the major protein in preparations of an acetylcholine receptor-inducing activity that has been purified greater than 10(6)-fold from brain on the basis of its ability to stimulate synthesis of nicotinic receptors by cultured myotubes. The ch-PrLP gene is expressed in the spinal cord and brain as early as embryonic day 6; and in the spinal cord, the protein appears to be concentrated in motor neurons. Our results therefore raise the possibility that prion proteins serve normally to regulate the chemoreceptor number at the neuromuscular junction and perhaps in the central nervous system as well.
Two nonallelic insulin genes in Xenopus laevis are expressed differentially during neurulation in prepancreatic embryos.
Authors: Shuldiner A R , de Pablo F , Moore C A , Roth J
Published: May 2006

Insulin, traditionally regarded as a metabolic hormone, also can potently stimulate growth and differentiation in many cell types. To study further the potential role of insulin during early embryogenesis, we have used the amphibian Xenopus laevis, a versatile model of vertebrate development. Using (i) nucleotide sequences of two previously cloned cDNAs that correspond to two different nonallelic Xenopus insulin genes (both of which are expressed in the adult pancreas) and (ii) a modification of the highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) method developed in our laboratory, designated RNA template-specific PCR (RS-PCR), we now find that mRNAs for both Xenopus insulins I and II are present in mature (stage VI) oocytes but not in less-mature oocytes (stages I and IV) or in unfertilized eggs. The Xenopus insulin II gene is differentially expressed during early neurulation (stage 13), while only the insulin I gene is expressed at stage 21, when the neural tube is closing and cephalization is beginning. During later stages (i.e., stage 26) there is a region in the head that appears to be transcribing only the insulin I gene, while mRNAs for both insulins I and II are present in the body region. These findings show that the two nonallelic insulin genes are expressed differentially in Xenopus embryos in a stage- and region-specific manner; because appropriate receptors are also present, we suggest a role for insulin during early nervous system development well before the emergence of pancreatic beta cells.
Highly divergent molecular variants of human T-lymphotropic virus type I from isolated populations in Papua New Guinea and the Solomon Islands.
Authors: Gessian A , Yanagihara R , Franchini G , Garruto R M , Jenkins C L , Ajdukiewicz A B , Gallo R C , Gajdusek D C
Published: May 2006

To determine the molecular genetic relationship between Melanesian strains of human T-lymphotropic virus type I (HTLV-I) and cosmopolitan prototype HTLV-I, we amplified by PCR, then cloned, and sequenced a 522-base-pair region of the HTLV-I env gene in DNA extracted from uncultured (fresh) and cultured peripheral blood mononuclear cells obtained from six seropositive Melanesian Papua New Guineans and Solomon Islanders, including a Solomon Islander with HTLV-I myeloneuropathy. Unlike isolates of HTLV-I from Japan, the West Indies, the Americas, and Africa, which share greater than or equal to 97% sequence homology, the Melanesian strains of HTLV-I were only 91.8%-92.5% identical with a prototype Japanese HTLV-IATK-1. The nucleotide sequence of proviral DNA from the Solomon Islander with HTLV-I myeloneuropathy also diverged markedly from that of HTLV-I isolated from Japanese patients with HTLV-I-associated myelopathy and from Jamaican patients with tropical spastic paraparesis, suggesting that these variant viruses are capable of causing disease. The HTLV-I variants from Papua New Guineans, in turn, differed by nearly 4% from the Melanesian variants from Solomon Islanders, indicating the existence of another HTLV-I quasi-species. By contrast, HTLV-I strains from two residents of Bellona Island, a Polynesian Outlier within the Solomon Islands, were closely related to cosmopolitan prototype HTLV-I (greater than or equal to 97% sequence identity), suggesting recent introduction, possibly during this century. These findings are consistent with a proto-Melanesian HTLV-I strain of archaic presence, which evolved independently of contemporary cosmopolitan strains, and pose new questions about the origin and global dissemination of HTLV-I.
Envelope proteins from clinical isolates of human immunodeficiency virus type 1 that are refractory to neutralization by soluble CD4 possess high affinity for the CD4 receptor.
Authors: Brighty D W , Rosenberg M , Chen I S , Ivey-Hoyle M
Published: May 2006

Recent evidence indicates that primary clinical isolates of human immunodeficiency virus type 1 (HIV-1) require significantly more soluble CD4 (sCD4) to block infection than the prototypic laboratory strain HTLV-IIIB. The currently accepted explanation for these observations is that the envelope glycoproteins from primary clinical isolates possess lower affinities for CD4 than laboratory strains. This observation has far reaching implications for the clinical effectiveness of sCD4. To test whether the resistance of clinical isolates to sCD4 neutralization correlates with low-affinity binding to gp120, we have compared gp120 glycoproteins derived from the clinical isolates HIV-1 JR-CSF and JR-FL with those derived from the prototypic strain HIV-1 BH10 in quantitative sCD4 binding studies. Surprisingly, our results demonstrate that gp120 derived from HIV-1 JR-CSF and JR-FL possess sCD4 binding affinities of equal or greater magnitude than gp120 derived from HIV-1 BH10. Thus primary clinical HIV-1 isolates can and do possess gp120 with high affinity for CD4, and sensitivity to neutralization by sCD4 is dependent upon factors other than the intrinsic affinity of gp120 for CD4.
Characterization of naturally processed antigen bound to major histocompatibility complex class II molecules.
Authors: Srinivasan M , Marsh E W , Pierce S K
Published: May 2006

Helper T lymphocytes recognize peptide fragments of antigen bound to major histocompatibility complex (MHC) class II molecules presented on the surface of antigen-presenting cells (APCs). Previous studies showed that the MHC class II, I-Ek molecules purified from APCs that had processed Drosophila melanogaster cytochrome c (DMc) contained functional, processed antigen-I-Ek complexes. This was demonstrated by the ability of purified I-Ek, incorporated into liposomes, to stimulate DMc-specific T cells in the absence of any additional antigen. Here we describe the isolation and characterization of the processed antigen bound to I-Ek. This was accomplished using DMc radiolabeled across its entire length by reductive methylation of its lysine residues, allowing an analysis of the totality of processed antigen bound to MHC class II molecules. After processing, only about 0.2% of the APC I-Ek molecules contained processed DMc (approximately 800 per cell), yet these were sufficient to stimulate specific T cells. The DMc peptides isolated from the I-Ek molecules showed only two predominant radioactive peaks as analyzed by reverse-phase chromatography. Less processed antigen was bound to purified I-Ak molecules, and these peptides were distinct from those bound to I-Ek. The association of processed DMc with the I-Ek and I-Ak molecules appears highly specific in that no radiolabeled peptides were isolated from purified MHC class I molecules, Kk and Dk, or from the B-cell differentiation antigen B220. The majority of processed antigen-I-Ek complexes migrated more slowly than the majority of the I-Ek protein as analyzed by SDS/PAGE under nonreducing conditions without heating of the sample. This form of I-Ek may be analogous to the earlier described "floppy" form of MHC class II molecules [Dormair, K., Rothenhausler, B. & McConnell, H. M. (1990) Cold Spring Harbor Symp. Quant. Biol. 54, 409-416]. Since newly processed antigen binds nearly exclusively to this slow-migrating form, it may be of functional significance.
Light-dependent channels from excised patches of Limulus ventral photoreceptors are opened by cGMP.
Authors: Bacigalupo J , Johnson E C , Vergara C , Lisman J E
Published: May 2006

The identity of the second messenger that directly activates the light-dependent conductance in invertebrate photoreceptors remains unclear; the available evidence provides some support for cGMP and Ca2+. To resolve this issue we have applied these second messengers to membrane patches excised from the light-sensitive lobe of Limulus ventral photoreceptors. Our results show that these patches contain channels that can be opened by cGMP, but not by Ca2+. These cGMP-activated channels closely resemble the channels activated by light in cell-attached patches. This evidence suggests that cGMP is the messenger that opens the light-dependent channel in invertebrate photoreceptors.
Identification of monoclonal antibody epitopes and critical residues for rhinovirus binding in domain 1 of intercellular adhesion molecule 1.
Authors: McClelland A , deBear J , Yost S C , Meyer A M , Marlor C W , Greve J M
Published: May 2006

Intercellular adhesion molecule 1 (ICAM-1) is the cellular receptor for the major group of human rhinoviruses (HRVs) and the adhesion ligand of lymphocyte function-associated antigen 1. Analysis of a series of chimeric exchanges between human and murine ICAM-1 shows that two distinct epitopes recognized by monoclonal antibodies that block rhinovirus attachment and cell adhesion map to the N-terminal first domain of ICAM-1. Furthermore the specificity for HRV binding is entirely contained within the first 88 amino acids. Mutagenesis of the four sites of N-linked glycosylation within the second domain shows that carbohydrate is not involved in virus recognition. Homologue replacement mutagenesis localizes the epitopes for virus-blocking antibodies to two regions of domain 1 predicted to form beta strand D and the loop between the F and G strands of an immunoglobulin-fold structure. Analysis of virus binding to the mutants predicts a large surface of contact between HRV and ICAM-1 domain 1 but shows that the regions most important for virus binding are coincident with the monoclonal antibody epitopes.
Molecular characterization of a region of DNA associated with mutations at the agouti locus in the mouse.
Authors: Bultman S J , Russell L B , Gutierrez-Espeleta G A , Woychik R P
Published: May 2006

Molecular characterization of a radiation-induced agouti (a)-locus mutation has resulted in the isolation of a segment of DNA that maps at or near the a locus on chromosome 2 in the mouse. This region of DNA is deleted in several radiation- or chemical-induced homozygous-lethal a-locus mutations and is associated with specific DNA structural alterations in two viable a-locus mutations. We propose that DNA probes from this region of chromosome 2 will be useful for ultimately characterizing the individual gene or genes associated with a-locus function.
Centromere formation in mouse cells cotransformed with human DNA and a dominant marker gene.
Authors: Hadlaczky G , Praznovszky T , Cserpán I , Keresö J , Péterfy M , Kelemen I , Atalay E , Szeles A , Szelei J , Tubak V
Published: May 2006

A 13,863-base-pair (bp) putative centromeric DNA fragment has been isolated from a human genomic library by using a probe obtained from metaphase chromosomes of human colon carcinoma cells. The abundance of this DNA was estimated to be 16-32 copies per genome. Cotransfection of mouse cells with this sequence and a selectable marker gene (aminoglycoside 3'-phosphotransferase type II, APH-II) resulted in a transformed cell line carrying an additional centromere in a dicentric chromosome. This centromere was capable of binding an anti-centromere antibody. In situ hybridization demonstrated that the human DNA sequence as well as the APH-II gene and vector DNA sequences were located only in the additional centromere of the dicentric chromosome. The extra centromere separated from the dicentric chromosome, forming a stable minichromosome. This functional centromere linked to a dominant selectable marker may be a step toward the construction of an artificial mammalian chromosome.