bluuebunny/crossref_metadata_2025 huggingface dataset:
T-cell receptor gene rearrangements in functional T-cell clones from severe combined immune deficient (scid) mice: reversion of the scid phenotype in individual lymphocyte progenitors.
Authors: Petrini J H , Carroll A M , Bosma M J
Published: May 2006

The severe combined immune deficient (scid) mouse mutant is characterized by a general absence of functional B and T lymphocytes. This deficiency appears to result from a defect in the variable-diversity-joining (VDJ) recombinase system, which is responsible for the assembly of V, D, and J gene segments that code for immunoglobulin and T-cell receptor (TCR) V regions. Most rearranged immunoglobulin or TCR genes in transformed scid lymphocytes contain abnormal J-associated deletions and are nonfunctional. A few functional lymphocyte clones do arise, however, in some young adult scid mice and in virtually all old scid mice; this phenomenon is referred to as leakiness. Alloreactive, CD3+ T-cell clones were isolated from leaky scid mice and the status of their TCR beta and gamma loci was examined in an effort to assess the nature of the recombinase activity that gives rise to functional scid lymphocytes. The recombination junctions of six gamma and two beta alleles were sequenced, representing four alloreactive T-cell clones. All of the junctions were indistinguishable from those seen in normal cells. These results cannot be attributed to selection by antigen because other rearranged TCR genes account for the TCR molecules expressed by these T-cell clones. We conclude that reversion of the scid phenotype can occur in rare lymphocyte progenitors and may account for most functional lymphocyte clones in leaky scid mice.
Low density lipoprotein receptor-binding activity in human tissues: quantitative importance of hepatic receptors and evidence for regulation of their expression in vivo.
Authors: Rudling M J , Reihnér E , Einarsson K , Ewerth S , Angelin B
Published: May 2006

The heparin-sensitive binding of 125I-labeled low-density lipoprotein (LDL) to homogenates from 18 different normal human tissues and some solid tumors was determined. The binding to adrenal and liver homogenates fulfilled criteria established for the binding of LDL to its receptor--namely, (i) saturability, (ii) sensitivity to proteolytic destruction, (iii) inhibition by EDTA, and (iv) heat sensitivity. When the binding of 125I-labeled LDL was assayed at a constant concentration (50 micrograms/ml), the adrenal gland and the ovary had the highest binding of normal tissues. The highest binding per g of tissue overall was obtained in homogenates of a gastric carcinoma and a parotid adenoma. When the weights of the parenchymatous organs were considered, the major amount of LDL receptors was contained in the liver. To study the possible regulation of hepatic LDL-receptor expression, 11 patients were pretreated with cholestyramine (8 g twice a day for 3 weeks). Increased binding activity (+105%, P less than 0.001) was obtained in homogenates from liver biopsies from the cholestyramine-treated patients as compared with 12 untreated controls. It is concluded that the liver is the most important organ for LDL catabolism in humans and that the receptor activity in this organ can be regulated upon pharmacologic intervention. Further studies are needed to confirm the possibility that certain solid tumors can exhibit high numbers of LDL receptors.
Conservation of the regulatory subunit for the Clp ATP-dependent protease in prokaryotes and eukaryotes.
Authors: Gottesman S , Squires C , Pichersky E , Carrington M , Hobbs M , Mattick J S , Dalrymple B , Kuramitsu H , Shiroza T , Foster T
Published: May 2006

Bacteria, tomatoes, and trypanosomes all contain genes for a large protein with extensive homology to the regulatory subunit, ClpA, of the ATP-dependent protease of Escherichia coli, Clp. All members of the family have between 756 and 926 amino acids and contain two large regions, of 233 and 192 amino acids, each containing consensus sequences for nucleotide binding. Within these regions there is at least 85% similarity between the most distant members of the family. The high degree of similarity among the ClpA-like proteins suggests that Clp-like proteases are likely to be important participants in energy-dependent proteolysis in prokaryotic and eukaryotic cells.
Metastability of the folded states of globular proteins.
Authors: Honeycutt J D , Thirumalai D
Published: May 2006

The possibility that several metastable minima exist in which the folded forms of a polypeptide chain have similar structural characteristics but different energies is suggested. The validity of this hypothesis is illustrated with the aid of simulation methods on a model protein that folds into a beta-barrel structure. Some implications of this hypothesis such as the existence of multiple pathways with intermediates for protein folding are discussed.
Hydrolysis of GTP by p21NRAS, the NRAS protooncogene product, is accompanied by a conformational change in the wild-type protein: use of a single fluorescent probe at the catalytic site.
Authors: Neal S E , Eccleston J F , Webb M R
Published: May 2006

2'(3')-O-(N-Methyl)anthraniloylguanosine 5'-triphosphate (mantGTP) is a fluorescent analogue of GTP that has similar properties to the physiological substrate in terms of its binding constant and the kinetics of its interactions with p21NRAS, the NRAS protooncogene product. There is a 3-fold increase in fluorescence intensity when mantGTP binds to p21NRAS. The rate constant for the cleavage of mantGTP complexed with the protein is similar to that of GTP, and cleavage is accompanied by a fluorescence intensity change in the wild-type protein complex. A two-phase fluorescence change also occurs when the nonhydrolyzable analogue 2'(3')-O-(N-methyl)anthraniloylguanosine 5'-[beta, gamma-imido]triphosphate (mantp[NH]ppG) binds to wild-type p21NRAS. The second phase occurs at the same rate as the second phase observed after mantGTP binding. Thus this second phase is probably a conformation change of the p21NRAS nucleotiside triphosphate complex and that the change controls the rate of GTP hydrolysis on the protein. With a transforming mutant, [Asp12]-p21NRAS, there is no second phase of the fluorescence change after mantGTP or mantp[NH]ppG binding, even though mantGTP is hydrolyzed. This shows that an equivalent conformational change does not occur and thus the mutant may stay in a "GTP-like" conformation throughout the GTPase cycle. These results are discussed in terms of the proposed role of p21NRAS in signal transduction and the transforming properties of the mutant.
Evidence for the evolutionary origin of human chromosome 21 from comparative gene mapping in the cow and mouse.
Authors: Threadgill D S , Kraus J P , Krawetz S A , Womack J E
Published: May 2006

To determine the extent of conservation between bovine syntenic group U10, human chromosome 21 (HSA 21), and mouse chromosome 16 (MMU 16), 11 genes were physically mapped by segregation analysis in a bovine-hamster hybrid somatic cell panel. The genes chosen for study span MMU 16 and represent virtually the entire q arm of HSA 21. Because the somatostatin gene (SST), an HSA 3/MMU 16 locus, was previously shown to be in U10, the transferrin gene (TF), an HSA 3/MMU 9 marker, was also mapped to determine whether U10 contains any HSA 3 genes not represented on MMU 16. With the exception of the protamine gene PRM1 (HSA 16/MMU 16), all of the genes studied were syntenic on bovine U10. Thus, all homologous loci from HSA 21 that have been studied in the cow are on a single chromosome. The bovine homolog of HSA 21 also carries several HSA 3 genes, two of which have homologous loci on MMU 16. The syntenic association of genes from the q arm of HSA 3 with HSA 21 genes in two mammalian species, the mouse and the cow, indicates that HSA 21 may have that contained genes now residing on HSA 3. Additionally, the syntenic association of TF with SST in the cow permits the prediction that the rhodopsin gene (RHO) is proximal to TF on HSA 3q.
Functional division and reconstruction of a plasmid replication origin: molecular dissection of the oriV of the broad-host-range plasmid RSF1010.
Authors: Honda Y , Sakai H , Hiasa H , Tanaka K , Komano T , Bagdasarian M
Published: May 2006

Two single-stranded DNA initiation signals (designated ssi) present in the origin of vegetative DNA replication (oriV) of the broad-host-range plasmid RSF1010 are essential for the priming of replication of each complementary DNA strand of this plasmid in Escherichia coli. Each of the RSF1010 ssi signals, ssiA and ssiB, could be replaced by a primosome assembly site from plasmid pACY184 or from bacteriophage phi X174. In these chimeric origins, replication of the strand complementary to that containing the primosome assembly site was no longer dependent on the RSF1010 primase, protein RepB', but required the E. coli primase, DnaG. If both ssiA and ssiB sites of RSF1010 were replaced by primosome assembly sites, protein RepB' was no longer essential for the replication at this origin, whereas proteins RepA and RepC of RSF1010 were still required. These results strongly suggest that the two ssi sites and the RepB' protein actually direct the priming of DNA synthesis in the replication of RSF1010, and the proteins RepA and RepC are involved in the prepriming events--i.e., the opening of the DNA duplex at oriV. It is evident that the origin of RSF1010 can be separated into three functional domains and reconstructed by replacing the ssi sites with heterologous elements.
Three adjacent binding sites for cAMP receptor protein are involved in the activation of the divergent malEp-malKp promoters.
Authors: Vidal-Ingigliardi D , Raibaud O
Published: May 2006

The divergent malEFG and malK-lamB-malM operons in Escherichia coli are controlled by partially overlapping promoters, whose activity depends on the presence of two transcriptional activators, MalT and the cAMP receptor protein (CRP). The 271-base-pair region separating the transcription start points of the promoters malEp and malKp comprises a compact array of binding sites for MalT and CRP. We report the characterization of the in vitro interactions of CRP with its four adjacent binding sites and the analysis of their function in vivo. By using the DNase I footprinting technique, we showed that CRP binds with high affinity to the three malEp-proximal sites and with a low affinity to the fourth site. CRP binding to these sites is not cooperative, even though they are adjacent and located on the same face of the DNA double helix. Each of these sites was destroyed by localized mutagenesis and the residual activity of the promoters was measured in vivo. Mutations in any of the three high-affinity binding sites reduced both malEp and malKp activity. The participation of several adjacent bound CRP molecules in the activation of a promoter is an unprecedented observation and might involve molecular mechanisms quite different from those used in the other CRP-controlled promoters.
NMR and molecular modeling evidence for a G.A mismatch base pair in a purine-rich DNA duplex.
Authors: Li Y , Zon G , Wilson W D
Published: May 2006

1H NMR experiments indicate that the oligomer 5'-d(ATGAGCGAATA) forms an unusual 10-base-pair duplex with 4 G.A base pairs (underlined) and a 3' unpaired adenosine. NMR results indicate that guanosine imino protons of the G.A mismatches are not hydrogen bonded but are stacked in the helix. A G----I substitution in either G.A base pair causes a dramatic decrease in duplex stability and indicates that hydrogen bonding of the guanosine amino group is critical. Nuclear Overhauser effect spectroscopy (NOESY) and two-dimensional correlated spectroscopy (COSY) results indicate that the overall duplex conformation is in the B-family. Cross-strand NOEs in two-dimensional NOESY spectra between a mismatched AH2 and an AH1' of the other mismatched base pair and between a mismatched GH8 and GNH1 of the other mismatch establish a purine-purine stacking pattern, adenosine over adenosine and guanosine over guanosine, which strongly stabilizes the duplex. A computer graphics molecular model of the unusual duplex was constructed with G.A base pairs containing A-NH2 to GN3 and G-NH2 to AN7 hydrogen bonds and B-form base pairs on both sides of the G.A pairs [5'-d(ATGAGC)]. The energy-minimized duplex satisfies all experimental constraints from NOESY and COSY results. A hydrogen bond from G-NH2 of the mismatch to a phosphate oxygen is predicted.
Extrathymic origin of intestinal intraepithelial lymphocytes bearing T-cell antigen receptor gamma delta.
Authors: Bandeira A , Itohara S , Bonneville M , Burlen-Defranoux O , Mota-Santos T , Coutinho A , Tonegawa S
Published: May 2006

The kinetics of postnatal intestinal colonization by T cells carrying gamma delta and alpha beta T-cell antigen receptors were studied in nude and normal mice by flow cytometry and immunohistology. Furthermore, gamma delta and alpha beta T-cell development was analyzed in lethally irradiated mice that were reconstituted by fetal liver precursors with or without a thymus. Our results establish that a major subpopulation of gamma delta intestinal intraepithelial lymphocytes is produced from uncommitted precursors at extrathymic sites. This work further shows that a small pool of T cells carrying alpha beta T-cell receptors can also differentiate extrathymically from CD3- fetal liver precursors but with rates of production and peripheral expansion much reduced as compared with those observed in thymus-bearing animals.
A mutagenesis study of a catalytic antibody.
Authors: Jackson D Y , Prudent J R , Baldwin E P , Schultz P G
Published: May 2006

We have generated seven site-specific mutations in the genes encoding the variable region of the heavy chain domain (VH) of the phosphocholine-binding antibody S107. S107 is a member of a family of well-characterized highly homologous antibodies that bind phosphorylcholine mono- and diesters. Two of these antibodies, MOPC-167 and T15, have previously been shown to catalyze the hydrolysis of 4-nitrophenyl N-trimethylammonioethyl carbonate. Two conserved heavy-chain residues, Tyr-33 and Arg-52, were postulated to be involved in binding and hydrolysis of 4-nitrophenylcholine carbonate esters. To more precisely define the catalytic roles of these residues, three Arg-52 mutants (R52K, R52Q, R52C) and four Tyr-33 mutants (Y33H, Y33F, Y33E, Y33D) of antibody S107 were generated. The genes encoding the VH binding domain of S107 were inserted into plasmid pUC-fl, and in vitro mutagenesis was performed. The wild-type and mutant S107 antibodies were expressed in P-3X63-Ag8.653 (P-3) myeloma cells by using a modified SV2 shuttle vector. The catalytic properties of wild-type antibody S107 are similar to those of the phosphocholine-specific antibody T15, which has the same VH protein sequence. In general, mutations at Tyr-33 had little effect on catalytic activity, whereas mutations at Arg-52 that result in loss of the positively charged side chain significantly lower the catalytic activity of S107. One mutant, Y33H, catalyzed the hydrolysis of 4-nitrophenyl N-trimethylammonioethyl carbonate with a kcat of 5.7 min-1 and a Km of 1.6 mM at pH 7.5. These results not only demonstrate the importance of electrostatic interactions in catalysis by antibody S107 but also show that catalytic side chains can be introduced into antibodies to enhance their catalytic efficiency.
Photoactivation of the 2-(methylthio)ethanesulfonic acid reductase from Methanobacterium.
Authors: Olson K D , McMahon C W , Wolfe R S
Published: May 2006

Inactive 2-(methylthio)ethanesulfonic acid (CH3-S-CoM) reductase was partially activated by exposure to light. This simplified system replaces the complex enzymatic system of protein components A2, A3a, A3b, and ATP, which previously represented the only available means of reactivating the enzyme. Components necessary for light activation include N-(7-mercaptoheptanoyl)-L-threonine O3-phosphate (HS-HTP), CH3-S-CoM, titanium(III) citrate [Ti(III)Cit], and light above 400 nm. Photoactivation was inhibited by known inhibitors of methanogenesis: 2-bromoethanesulfonate (BES), N-(6-mercaptohexanoyl)-L-threonine O3-phosphate, N-(8-mercaptooctanoyl)-L-threonine O3-phosphate, and sodium dithionite. Methanogenesis continued when the light-activated reaction mixture was incubated in the dark. Although the specific activity was low (35 nmol of CH4 per h per mg of protein) the reaction products methane and the unsymmetrical disulfide of 2-mercaptoethanesulfonate (HS-CoM) and HS-HTP were identified. We were unable to photoactivate a reaction mixture containing the isolated prosthetic group, native F430, or its analogues.
Reshaping a therapeutic CD4 antibody.
Authors: Gorman S D , Clark M R , Routledge E G , Cobbold S P , Waldmann H
Published: May 2006

An immunosuppressive rat antibody (Campath-9) against human CD4 has been reshaped for use in the management of autoimmunity and the prevention of graft rejection. Two different forms of the reshaped antibody were produced that derive their heavy chain variable region framework sequences from the human myeloma proteins KOL or NEW. When compared to a chimeric form of the CD4 antibody, the avidity of the KOL-based reshaped antibody was only slightly reduced, whereas that of the NEW-based reshaped antibody was very poor. The successful reshaping to the KOL-based framework was by a procedure involving the grafting of human framework sequences onto the cloned rodent variable region by in vitro mutagenesis.
Mixed inhibitors of angiotensin-converting enzyme (EC 3.4.15.1) and enkephalinase (EC 3.4.24.11): rational design, properties, and potential cardiovascular applications of glycopril and alatriopril.
Authors: Gros C , Noël N , Souque A , Schwartz J C , Danvy D , Plaquevent J C , Duhamel L , Duhamel P , Lecomte J M , Bralet J
Published: May 2006

Angiotensin-converting enzyme (ACE) and enkephalinase, two cell surface metallopeptidases, are responsible for angiotensin II formation and atrial natriuretic factor (ANF) degradation, respectively, and thereby play a critical role in the metabolism of hormonal peptides exerting essentially opposite actions in cardiovascular regulations. To affect simultaneously both hormonal systems by a single molecular structure, we have designed glycoprilat and alatrioprilat [(S)-N-[3-(3,4-methylene-dioxyphenyl)-2-(mercaptomethyl)-1-oxoprop yl] glycine and -alanine, respectively]. In vitro the two compounds inhibit both ACE and enkephalinase activities with similar, nanomolar potencies, and in vivo, glycopril and alatriopril, the corresponding diester prodrugs, occupy the two enzyme molecules in lung at similar low dosages (0.2-0.5 mg/kg of body weight, per os). The high potency of these compounds is attributable to interaction of the methylenedioxy group with the S1 subsite of ACE and of the aromatic ring with the S1' subsite of enkephalinase. In rodents, low doses of these mixed inhibitors exert typical actions of ACE inhibitors--i.e., prevention of angiotensin I-induced hypertension--as well as of enkephalinase inhibitors--i.e., protection from 125I-ANF degradation or enhancement of diuresis and natriuresis following acute extracellular volume expansion. In view of the known counterbalanced physiological actions of the two hormonal peptides, whose metabolism is controlled by ACE and enkephalinase, mixed inhibitors of the two peptidases show promise for the treatment of various cardiovascular and salt-retention disorders.
Crystal structure of a bovine neurophysin II dipeptide complex at 2.8 A determined from the single-wavelength anomalous scattering signal of an incorporated iodine atom.
Authors: Chen L Q , Rose J P , Breslow E , Yang D , Chang W R , Furey W F , Sax M , Wang B C
Published: May 2006

The crystal structure of a dipeptide complex of bovine neurophysin II has been solved at 2.8 A resolution solely by using single-wavelength anomalous scattering data from a single iodinated derivative. The asymmetric unit is an elongated tetramer of dimensions 110 x 40 x 30 A, composed of two dimers related by pseudo twofold symmetry. Each monomer consists of two homologous layers, each with four antiparallel beta-strands. The two regions are connected by a helix followed by a long loop. Monomer-monomer contacts involve antiparallel beta-sheet interactions, which form a dimer with two layers of eight beta-strands. One peptide per monomer occupies the principal hormone-binding pocket formed by part of the amino-terminal region and parts of the connecting helix and loop, with binding to protein consistent with conclusions drawn from solution studies. Dimer-dimer contacts involve the Tyr49 region adjacent to this site. A fifth dipeptide, of unknown biological significance, helps to stabilize one of the monomer-monomer interfaces and the tetramer-tetramer network in the crystal.
Expression of growth/differentiation factor 1 in the nervous system: conservation of a bicistronic structure.
Authors: Lee S J
Published: May 2006

Growth/differentiation factor 1 (GDF-1) is a recently described member of the transforming growth factor beta superfamily isolated from a day-8.5 mouse embryo cDNA library. Northern (RNA) analysis of embryonic mRNA detected two GDF-1 transcripts [1.4 kilobases (kb) and 3.0 kb in length] displaying distinct temporal patterns of expression. Only the 3.0-kb transcript was detected in adult tissues, where its expression was restricted almost exclusively to the central nervous system. Comparison of murine and human brain cDNA sequences corresponding to the 3.0-kb transcript revealed high conservation of two nonoverlapping open reading frames with poor conservation of the intervening spacer region and the putative 5' and 3' untranslated sequences. By immunohistochemical analysis, the protein encoded by the downstream open reading frame (GDF-1) was detected exclusively in the brain, spinal cord, and peripheral nerves in day-14.5 mouse embryos. The upstream open reading frame encodes a protein of unknown function containing multiple putative membrane-spanning domains. These findings raise the possibility that this mRNA may give rise to two different proteins.
Ion channels in transit: voltage-gated Na and K channels in axoplasmic organelles of the squid Loligo pealei.
Authors: Wonderlin W F , French R J
Published: May 2006

Ion channels that give rise to the excitable properties of the neuronal plasma membrane are synthesized, transported, and degraded in cytoplasmic organelles. To determine whether plasma membrane ion channels from these organelles could be physiologically activated, we extruded axoplasm from squid giant axons, dissociated organelles from the cytoskeletal matrix, and fused the free organelles with planar lipid bilayers. Three classes of ion channels normally associated with the plasma membrane were identified based on conductance, selectivity, and gating properties determined from steady-state single-channel recordings: (i) voltage-dependent Na channels, (ii) voltage-dependent delayed rectifier K channels, and (iii) large, voltage-independent K channels. The identity of the delayed rectifier channels was confirmed by reconstructing the time course of activation from single-channel responses to depolarizing voltage steps applied across the bilayer. These observations suggest that several classes of plasma membrane ion channels are transported in cytoplasmic organelles in physiologically active forms.
Different functional boundaries for the major antigenic region of two cytochromes c.
Authors: Jemmerson R , Johnson J G
Published: May 2006

The antigenic sites of horse and rat cytochromes c in the major antigenic region were compared using a panel of variant cytochromes c and a large number of BALB/c mouse monoclonal antibodies (mAbs) in competitive ELISAs. The major antigenic region of cytochrome c is located on the surface opposite of that containing the exposed heme crevice. mAbs specific for this region on rat cytochrome c were affected in binding by amino acid substitutions at positions 62 and at one or more of positions 3, 100, 103, and 104 but not by a substitution at position 89. In contrast, mAbs specific for the same region on horse cytochrome c were affected by amino acid substitutions at positions 62, (probably) 60, and 89. Some, but not all, of the anti-horse cytochrome c mAbs were affected by amino acid substitutions at one or more of positions 3, 100, 103, and 104. Thus, the functional boundaries of this antigenic region are different for these two cytochromes c, as shown primarily by the differential effects of residue 89. This distinction is most likely due to one or more of the four amino acid differences between horse and rat cytochromes c on the antigenic surface that result in different physicochemical properties for the horse and rat proteins. The results suggest that as yet unidentified physicochemical parameters, possibly surface topography and/or charge distribution, influence the focusing of antibodies onto the surface of a protein antigen.
A more biologically plausible learning rule for neural networks.
Authors: Mazzoni P , Andersen R A , Jordan M I
Published: May 2006

Many recent studies have used artificial neural network algorithms to model how the brain might process information. However, back-propagation learning, the method that is generally used to train these networks, is distinctly "unbiological." We describe here a more biologically plausible learning rule, using reinforcement learning, which we have applied to the problem of how area 7a in the posterior parietal cortex of monkeys might represent visual space in head-centered coordinates. The network behaves similarly to networks trained by using back-propagation and to neurons recorded in area 7a. These results show that a neural network does not require back propagation to acquire biologically interesting properties.
Immunogenicity of an engineered internal image antibody.
Authors: Billetta R , Hollingdale M R , Zanetti M
Published: May 2006

We engineered an antibody expressing in the third complementarity-determining region of its heavy chain variable region a "foreign" epitope, the repetitive tetrapeptide Asn-Ala-Asn-Pro (NANP) of the circumsporozoite protein of Plasmodium falciparum parasite, one of the etiologic agents of malaria in humans. A monoclonal antibody to P. falciparum specific for the (NANP)n amino acid sequence bound to the engineered antibody, and a synthetic (NANP)3 peptide blocked this interaction. Immunization of rabbits and mice with the engineered antibody resulted in the elicitation of a humoral response to (NANP)3 synthetic peptide and P. falciparum parasite. In mice, in which immunity to the (NANP)n epitope is highly restricted by immune response genes, antibodies were induced in responder and nonresponder haplotypes of the major histocompatibility complex. Rabbit antibodies efficiently inhibited the in vitro invasion of cultured liver cells by P. falciparum parasite. Collectively, this study indicates that immunity to malaria in the absence of the parasite can be induced using antibody variable regions engineered to mimic the parasite's molecular structure. In general terms, the results suggest that antibody (idiotype) mimicry of an exogenous antigen is possible and may only require a discrete stretch of identity between the two molecules. The implication for the preparation of antibody-based vaccines and idiotype regulation of immunity are discussed.
Purification of murine suppressive factor of allergy into distinct CD23-modulating and IgE-suppressive proteins.
Authors: Matsushita S , Marcelletti J F , Katz L R , Katz D H
Published: May 2006

The murine suppressive factor of allergy (SFA) has been purified from a T-cell hybridoma and found to consist of two functionally and biochemically distinct protein molecules. One protein (17 kDa) modulates the low-affinity Fc receptor for IgE on lymphocytes (i.e., CD23); it decreases the binding avidity of IgE to CD23-bearing B cells without affecting quantitative expression of CD23 and is thus designated epsilon-receptor-modulating protein. The second protein (30 kDa) suppresses IgE biosynthesis (i.e., SFA). This purified SFA suppresses interleukin 4-induced IgE and IgG1 synthesis by lipopolysaccharide-activated spleen cells but has no effect on other antibody isotypes; since the activity of SFA is not blocked by anti-interferon gamma monoclonal antibody, it is thus distinct from interferon gamma. The data presented indicate that epsilon-receptor-modulating protein and SFA are protein molecules that are involved in modulating the CD23 molecule and IgE antibody synthesis, respectively.
Structural femtochemistry: experimental methodology.
Authors: Williamson J C , Zewail A H
Published: May 2006

The experimental methodology for structural femtochemistry of reactions is considered. With the extension of femtosecond transition-state spectroscopy to the diffraction regime, it is possible to obtain in a general way the trajectories of chemical reactions (change of internuclear separations with time) on the femtosecond time scale. This method, considered here for simple alkali halide dissociation, promises many applications to more complex reactions and to conformational changes. Alignment on the time scale of the experiments is also discussed.
Hyperalgesia induced in the rat by the amino-terminal octapeptide of nerve growth factor.
Authors: Taiwo Y O , Levine J D , Burch R M , Woo J E , Mobley W C
Published: May 2006

Nerve growth factor (NGF) in the mouse submandibular gland undergoes cleavage of its amino-terminal octapeptide when salivation is induced by epinephrine. The significance of this event is uncertain; cleaved NGF demonstrates bioactivity and no function has been attributed to the octapeptide produced (NGF-OP; Ser-Ser-Thr-His-Pro-Val-Phe-His). Enzyme inhibition studies indicating structural relatedness of NGF-OP and bradykinin (BK) prompted us to determine whether NGF-OP would elicit BK-like actions. We found that like BK, NGF-OP induced a decrease in mechanical nociceptive threshold (i.e., produced hyperalgesia) in the hairy skin of the rat. This effect was dose-dependent and sequence-specific; like BK it was attenuated by sympathectomy and indomethacin pretreatment. However, NGF-OP actions appeared to be distinct from those for BK in that tissue injury was required for NGF-OP to induce hyperalgesia. Furthermore, we found no evidence that NGF-OP bound to or activated BK receptors. Our data indicate that NGF-OP is a distinct mediator of hyperalgesia. We suggest that NGF-OP alters pain threshold in the injured target regions of NGF-responsive neurons.
In vivo random beta-glucuronidase gene fusions in Arabidopsis thaliana.
Authors: Kertbundit S , De Greve H , Deboeck F , Van Montagu M , Hernalsteens J P
Published: May 2006

Vectors were constructed for the isolation of random transcriptional and translational beta-glucuronidase gene fusions in plants. This system is based on the random integration of the transferred DNA (T-DNA) into the plant nuclear genome. The Escherichia coli beta-glucuronidase coding sequence without promoter, and also devoid of its ATG initiation site in the translational gene fusion vector, was inserted in the T-DNA with its 5' end at a distance of 4 base pairs from the right T-DNA border sequence. Transgenic plants can be selected by using a chimeric (P35S-nptII-3' ocs) kanamycin-resistance gene present in the same T-DNA. Subsequent screening of these for beta-glucuronidase expression allows the identification of clones harboring a fusion of the beta-glucuronidase coding sequence with plant 5' regulatory sequences. After transformation of Arabidopsis thaliana C24 root explants, beta-glucuronidase expression was detected in 54% and 1.6% of the plants transformed with the transcriptional and translational fusion vectors, respectively. Several different patterns of tissue-specific beta-glucuronidase expression were identified. The plant upstream sequence of a beta-glucuronidase fusion that is specifically expressed in the phloem of all organs was cloned and sequenced. After introduction in A. thaliana C24 and Nicotiana tabacum SR1, this sequence mediates the same highly phloem-specific beta-glucuronidase expression pattern as in the original transgenic plant from which it was isolated. These data demonstrate that this system facilitates the isolation and analysis of plant DNA sequences mediating regulated gene expression.
Newly synthesized RNA: simultaneous measurement in intact cells of transcription rates and RNA stability of insulin-like growth factor I, actin, and albumin in growth hormone-stimulated hepatocytes.
Authors: Johnson T R , Rudin S D , Blossey B K , Ilan J , Ilan J
Published: May 2006

The levels of several RNA transcripts in cultured hepatocytes are regulated by transcriptional and post-transcriptional mechanisms and are affected by growth hormone and insulin. We assessed the effects of these hormones on transcription rates and the stability of insulin-like growth factor I, actin, and albumin transcripts in intact cells of primary cultures of rat hepatocytes by analyzing thiol-labeled, newly synthesized RNA isolated by mercurated agarose affinity chromatography. The application of this concept to the measurement of transcript stability is presented in detail. The data indicate that growth hormone stimulates the transcription rates of insulin-like growth factor I, actin, and albumin genes. The stability of all three transcripts, particularly albumin, appears to be lower in growth hormone-containing medium than it is in insulin-containing medium. The experiments indicate that the rates of transcription and/or degradation of albumin mRNA are influenced by hormonal treatment. However, the cells maintain roughly constant albumin transcript levels independent of hormone treatment by compensatory changes in the rates of transcription and degradation.
A protein geranylgeranyltransferase from bovine brain: implications for protein prenylation specificity.
Authors: Yokoyama K , Goodwin G W , Ghomashchi F , Glomset J A , Gelb M H
Published: May 2006

A protein geranylgeranyltransferase (PGT) that catalyzes the transfer of a 20-carbon prenyl group from geranylgeranyl pyrophosphate to a cysteine residue in protein and peptide acceptors was detected in bovine brain cytosol and partially purified. The enzyme was shown to be distinct from a previously characterized protein farnesyltransferase (PFT). The PGT selectively geranylgeranylated a synthetic peptide corresponding to the C terminus of the gamma 6 subunit of bovine brain G proteins, which have previously been shown to contain a 20-carbon prenyl modification. Likewise, a peptide corresponding to the C terminus of human lamin B, a known farnesylated protein, selectively served as a substrate for farnesylation by the PFT. However, with high concentrations of peptide acceptors, both prenyl transferases were able to use either peptide as substrates and the PGT was able to catalyze farnesyl transfer. Among the prenyl acceptors tested, peptides and proteins with leucine or phenylalanine at their C termini served as geranylgeranyl acceptors, whereas those with C-terminal serine were preferentially farnesylated. These results suggest that the C-terminal amino acid is an important structural determinant in controlling the specificity of protein prenylation.
Crystal structure of [Leu1]zervamicin, a membrane ion-channel peptide: implications for gating mechanisms.
Authors: Karle I L , Flippen-Anderson J L , Agarwalla S , Balaram P
Published: May 2006

Structures in four different crystal forms of [Leu1]zervamicin (zervamicin Z-L, Ac-Leu-Ile-Gln-Iva-Ile5-Thr-Aib-Leu-Aib-Hyp10-Gln-Aib-Hyp-Aib-P ro15-Phol, where Iva is isovaline, Aib is alpha-amino isobutyric acid, Hyp is 4-hydroxyproline, and Phol is phenylalaninol), a membrane channel-forming polypeptide from Emericellopsis salmosynnemata, have been determined by x-ray diffraction. The helical structure is amphiphilic with all the polar moieties on the convex side of the bent helix. Helices are bent at Hyp10 from approximately 30 degrees to approximately 45 degrees in the different crystal forms. In all crystal forms, the peptide helices aggregate in a similar fashion to form water channels that are interrupted by hydrogen bonds between N epsilon H(Gln11) and O delta (Hyp10) of adjacent helices. The Gln11 side chain is folded in an unusual fashion in order to close the channel. Space is available for an extended conformation for Gln11, in which case the channel would be open, suggesting a gating mechanism for cation transport. Structural details are presented for one crystal form derived from methanol/water solution: C85H140N18O22. 10H2O, space group P21, a = 23.068(6) A, b = 9.162(3) A, c = 26.727(9) A, beta = 108.69(2) degrees (standard deviation of last digit is given in parentheses); overall agreement factor R = 10.1% for 5322 observed relfections [magnitude of Fo greater than 3 sigma (F)]; resolution, 0.93 A.
Rapid induction of heme oxygenase 1 mRNA and protein by hyperthermia in rat brain: heme oxygenase 2 is not a heat shock protein.
Authors: Ewing J F , Maines M D
Published: May 2006

Catalytic activity of heme oxygenase (heme, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.3) isozymes, HO-1 and HO-2, permits production of physiologic isomers of bile pigments. In turn, bile pigments biliverdin and bilirubin are effective antioxidants in biological systems. In the rat brain we have identified only the HO-1 isozyme of heme oxygenase as a heat shock protein and defined hyperthermia as a stimulus that causes an increase in brain HO-1 protein. Exposure of male rats to 42 degrees C for 20 min caused a rapid and marked increase in brain 1.8-kilobase HO-1 mRNA. Specifically, a 33-fold increase in brain HO-1 mRNA was observed within 1 h and sustained for at least 6 h posttreatment. In contrast, the two HO-2 homologous transcripts (1.3 and 1.9 kilobases) did not respond to heat shock; neither the ratio nor the level of the two messages differed from that of the control when measured either at 1, 6, or 24 h after hyperthermia. The induction of a 1.8-kilobase HO-1 mRNA resulted in a pronounced increase in HO-1 protein 6 h after hyperthermia, as detected by both Western immunoblot and RIA. Immunocytochemistry of rat brain showed discrete localization of HO-1-like protein only in neurons of select brain regions. Six hours after heat shock, an intense increase in HO-1-like protein was observed in both Purkinje cells of the cerebellum and epithelial cells lining the cerebral aqueduct of the brain. We suggest that the increase in HO-1 protein, hence increased capacity to form bile pigments, represents a neuronal defense mechanism against heat shock stress.
Molecular sequence accuracy and the analysis of protein coding regions.
Authors: States D J , Botstein D
Published: May 2006

Molecular sequences, like all experimental data, have finite error rates. The impact of errors on the information content of molecular sequence data is dependent on the analytic paradigm used to interpret the data. We studied the impact of nucleic acid sequence errors on the ability to align predicted amino acid sequences with the sequences of related proteins. We found that with a simultaneous translation and alignment algorithm, identification of sequence homologies is resilient to the introduction of random errors. Proteins with greater than 30% sequence identity can be reliably recognized even in the presence of 1% frameshifting (insertion or deletion) error rates and 5% base substitution rates. Incorporation of prior knowledge about the location and characteristics of errors improves tolerance to error of amino acid sequence alignments. Similarly, inclusion of prior knowledge of biased codon utilization by yeast (Saccharomyces cerevisiae) allows reliable detection of correct reading frames in yeast sequences even in the presence of 5% substitution and 1% frameshift errors.
Primary structure of a calcium channel that is highly expressed in the rat cerebellum.
Authors: Starr T V , Prystay W , Snutch T P
Published: May 2006

Previous molecular cloning experiments showed that multiple isoforms of the alpha 1 subunit of voltage-gated Ca channels are expressed in the mammalian brain (designated rbA, rbB, rbC, and rbD). We report here the isolation and characterization of cDNAs encoding the rat brain class A (rbA) Ca channel. The rbA-126 cDNA encodes a 2212-amino acid protein that shares 33% sequence identity with the alpha 1 subunits of cardiac and skeletal muscle dihydropyridine-sensitive Ca channels. When compared with other Ca channels, the rbA channel is notably different in both the carboxyl terminus and in a large (474-amino acid) hydrophilic segment between domains II and III. Northern blot analysis shows that rbA transcripts are expressed in all regions of the rat central nervous system, but most prominently in the cerebellum. A more widespread distribution of rbA Ca channels is indicated by PCR analysis, which demonstrates the presence of class A transcripts in the rat heart and pituitary but not in the spleen, kidney, or liver. The rbA cDNA appears to encode a Ca channel alpha 1 subunit that is distinct from the dihydropyridine-sensitive Ca channel sequences and that is expressed in a variety of excitable cells.
Interaction of hsp70 with unfolded proteins: effects of temperature and nucleotides on the kinetics of binding.
Authors: Palleros D R , Welch W J , Fink A L
Published: May 2006

Circular dichroism and HPLC gel filtration were used to show that cytosolic hsp70 is thermally stable but undergoes a conformational transition (midpoint, 43 degrees C; 57 degrees C in the presence of ATP or ADP) leading to oligomerization. hsp70 binds to unfolded, but not to folded, proteins in a temperature-dependent manner; complex formation is significant only at physiologically relevant temperatures. hsp70 binds ADP more tightly than ATP to form a binary complex, which binds to the unfolded protein more rapidly than free hsp70. ADP also inhibits the ATP-induced dissociation of the hsp70-protein complex. A regulatory role for the hsp70-nucleotide binary complexes is proposed.
Generation of a nested series of interstitial deletions in yeast artificial chromosomes carrying human DNA.
Authors: Campbell C , Gulati R , Nandi A K , Floy K , Hieter P , Kucherlapati R S
Published: May 2006

We have generated a nested series of interstitial deletions in a fragment of human X chromosome-derived DNA cloned into a yeast artificial chromosome (YAC) vector. A yeast strain carrying the YAC was transformed with a linear recombination substrate containing at one end a sequence that is uniquely represented on the YAC and at the other end a truncated long interspersed repetitive element (LINE 1, or L1). Homologous recombination between the YAC and the input DNA resulted in a nested series of interstitial deletions, the largest of which was 500 kilobases. In combination with terminal deletions that can be generated through homologous recombination, the interstitial deletions are useful for mapping and studying gene structure-function relationships.
Direct transport of ADPglucose by an adenylate translocator is linked to starch biosynthesis in amyloplasts.
Authors: Pozueta-Romero J , Frehner M , Viale A M , Akazawa T
Published: May 2006

Starch biosynthesis has been studied by using amyloplasts isolated from cultured cells of sycamore trees (Acer pseudoplatanus L.). Highly purified intact amyloplasts, free from mitochondria and starch granules derived from broken amyloplasts, were isolated from a Percoll step gradient. Subsequently, the double silicone oil layer centrifugation technique was used to study adenylate transport in the amyloplasts. An adenylate-specific carrier was found to be active in the uptake of ATP, ADP, AMP, and most importantly, ADPglucose (ADP-Glc). Kinetic analyses showed that the uptake of these adenylates was mutually competitive with each other. In contrast to the mitochondrial adenylate carrier, in amyloplasts only ATP and ADP-Glc uptake were inhibited by carboxyatractyloside. Evidence is presented that the ADP-Glc transported into the amyloplast stroma can be used in starch synthesis catalyzed by starch synthase (ADP-Glc:1,4-alpha-D-glucan 4-alpha-D-glucosyltransferase, EC 2.4.1.21). We propose that starch biosynthesis in amyloplasts is tightly coupled with the direct transport of ADP-Glc synthesized in the cytosol by sucrose synthase (ADP-Glc:D-fructose 2-alpha-D-glucosyltransferase, EC 2.4.1.13)
Signal convergence on protein kinase A as a molecular correlate of learning.
Authors: Aszódi A , Müller U , Friedrich P , Spatz H C
Published: May 2006

The response of a reaction network composed of protein kinase A, calpain, and protein phosphatase to transient cAMP and Ca2+ signals was studied. An essential feature of signal convergence is that the regulatory subunit of cAMP-dissociated protein kinase A undergoes limited proteolysis by the Ca(2+)-activated proteinase calpain. A dynamic model of this system based on kinetic differential equations was built and simulated by computer. The system shows analogies to typical features of associative learning such as acquisition, contiguity detection, extinction, and memory decay, suggesting that these biochemical reactions may be part of the molecular mechanism of learning in Drosophila.
Mechanisms of plasma membrane protein degradation: recycling proteins are degraded more rapidly than those confined to the cell surface.
Authors: Hare J F , Taylor K
Published: May 2006

Plasma membrane proteins of intact mouse 3T3 fibroblasts and H4-II-E-C3 hepatoma cells were separated into two groups based on their compartmentation between the cell surface and an intracellular compartment accessible at 20 degrees C but not at 0 degrees C. One group was derivatized at 0 degrees C with sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate but not at 20 degrees C. The second group was derivatized at 20 degrees C as well as at 0 degrees C. Derivatized proteins were isolated from 35S-labeled cells on streptavidin/agarose and resolved by two-dimensional PAGE. With few exceptions, pulse-chase experiments revealed that those proteins confined exclusively to the cell surface turned over slowly (t1/2, greater than 75 h), while those bimodally compartmentalized between the cell surface and the 20 degrees C accessible compartment were degraded more rapidly (t1/2, less than 31 h). These observations suggest a mechanism to explain the varied metabolic stability of plasma membrane proteins in which the half-life of each protein is determined by the proportion of time spent in the endocytic compartment.
Crosslinking of hemin to a specific site on the 90-kDa ferritin repressor protein.
Authors: Lin J J , Patino M M , Gaffield L , Walden W E , Smith A , Thach R E
Published: May 2006

Incubation of a 90-kDa ferritin repressor protein (FRP) with small amounts of radiolabeled hemin resulted in the formation of a strong interaction between the two that was stable to SDS/PAGE. (We refer to this interaction as a "crosslink," without intending to imply knowledge as to its chemical nature.) Of seven other proteins tested individually, only apohemopexin and bovine serum albumin showed similar crosslinking ability, albeit to a much lower extent. [14C]Hemin specifically crosslinked to FRP in the presence of a 50-fold excess of total wheat germ proteins. Inclusion of catalase did not prevent the reaction of hemin with FRP, suggesting that H2O2 is not involved. The subsequent addition of a stoichiometric amount of apohemopexin did not reverse the reaction. Exhaustive digestion of the complex with Staphylococcus aureus V8 protease produced a major labeled peptide of 17 kDa. These results show the existence of a highly specific, uniquely reactive hemin binding site on FRP.