bluuebunny/crossref_metadata_2025 huggingface dataset:
Human high molecular weight melanoma-associated antigen (HMW-MAA) mimicry by mouse anti-idiotypic monoclonal antibody MK2-23: induction of humoral anti-HMW-MAA immunity and prolongation of survival in patients with stage IV melanoma.
Authors: Mittelman A , Chen Z J , Yang H , Wong G Y , Ferrone S
Published: May 2006

Twenty-five patients with stage IV melanoma were immunized with the mouse anti-idiotypic monoclonal antibody (mAb) MK2-23 (2 mg per injection), which bears the internal image of the determinant defined by anti-HMW-MAA mAb 763.74. Two patients were inevaluable, since they did not complete 4 weeks of therapy. Only 14 patients developed antibodies that were shown by serological and immunochemical assays to recognize the same or spatially close determinant as the anti-HMW-MAA mAb 763.74 and to express the idiotope defined by mAb MK2-23 in their antigen-combining sites. Side effects that are likely to be caused by bacillus Calmette-Guérin present in the immunogen consisted of erythema, induration, and ulceration at the sites of the injections. Occasionally, patients complained of flu-like symptoms, arthralgias, and myalgias. Three of the patients who developed anti-HMW-MAA antibodies achieved a partial response. It consisted of a decrease in the size of metastatic lesions and lasted 52 weeks in 1 patient and 93 weeks in the other 2 patients. Survival of the 14 patients who developed anti-HMW-MAA antibodies was significantly (P = 0.0003) longer than that of the 9 patients without detectable humoral anti-HMW-MAA immunity development. In the multivariate analysis, such an association between development of anti-HMW-MAA antibodies and survival prolongation was still significant (P = 0.001) after adjustment for difference in performance status, the only confounding factor found to be significantly related to survival. Lastly, a significant (P = 0.03 by likelihood ratio test) interaction between anti-HMW-MAA antibodies and patients' performance status was found, since the prolongation of survival associated with anti-HMW-MAA antibodies was more marked in patients with a performance status of less than or equal to 70% than in those with a higher one. These results suggest that anti-idiotypic mAb MK2-23 may represent a useful immunogen to implement active specific immunotherapy in patients with melanoma.
Crystal structure of an Okazaki fragment at 2-A resolution.
Authors: Egli M , Usman N , Zhang S G , Rich A
Published: May 2006

In DNA replication, Okazaki fragments are formed as double-stranded intermediates during synthesis of the lagging strand. They are composed of the growing DNA strand primed by RNA and the template strand. The DNA oligonucleotide d(GGGTATACGC) and the chimeric RNA-DNA oligonucleotide r(GCG)d(TATACCC) were combined to form a synthetic Okazaki fragment and its three-dimensional structure was determined by x-ray crystallography. The fragment adopts an overall A-type conformation with 11 residues per turn. Although the base-pair geometry, particularly in the central TATA part, is distorted, there is no evidence for a transition from the A- to the B-type conformation at the junction between RNA.DNA hybrid and DNA duplex. The RNA trimer may, therefore, lock the complete fragment in an A-type conformation.
Mutational analysis of the integrase protein of human immunodeficiency virus type 2.
Authors: van Gent D C , Groeneger A A , Plasterk R H
Published: May 2006

Purified integrase protein (IN) can nick linear viral DNA at a specific site near the ends and integrate nicked viral DNA into target DNA. We have made a series of 43 site-directed point mutants of human immunodeficiency virus type 2 IN and assayed purified mutant proteins for the following activities: site-specific cleavage of viral DNA (donor cut), integration (strand transfer), and disintegration. In general, the different activities were similarly affected by the mutations. We found three mutations that (almost) totally abolished IN function: Asp-64-->Val, Asp-116-->Ile, and Glu-152-->Leu, whereas 25 mutations did not affect IN function. A few mutations affected the different activities differentially. Near the amino terminus a zinc finger-like sequence motif His-Xaa3-His-Xaa20-30-Cys-Xaa2-Cys is present in all retroviral IN proteins. Two mutations in this region (His-12-->Leu and Cys-40-->Ser) strongly inhibited donor cut but had less effect on strand transfer. The central region of IN is most highly conserved between retroviral INs. Three mutants in this region (Asn-117-->Ile, Asn-120-->Leu, and Lys-159-->Val) were inhibited in strand transfer but were inhibited less strongly in donor cut. Mutation of Asn-120 (to glycine, leucine, or glutamate) resulted in changes in integration-site preference, suggesting that Asn-120 is involved in interactions with target DNA. We did not find a mutant in which one activity was lost and the others were unaffected, supporting the notion that IN has only one active site for the catalysis of donor cut and strand transfer.
Detection of transcripts for the receptor for macrophage colony-stimulating factor, c-fms, in murine osteoclasts.
Authors: Hofstetter W , Wetterwald A , Cecchini M C , Felix R , Fleisch H , Mueller C
Published: May 2006

Macrophage colony-stimulating factor (M-CSF), whose action is restricted to the cell populations of the mononuclear phagocyte system, has recently been found to be required for osteoclastogenesis and bone resorption. To investigate the cells involved in the action of M-CSF in these processes, expression of c-fms mRNA, encoding the M-CSF receptor, was studied by in situ hybridization. Paws from murine embryos and newborn mice, tibiae from 2-day-old animals, as well as isolated osteoclasts, were hybridized with a c-fms-specific RNA probe. In bone, c-fms mRNA was detected only in cells at the late stages of osteoclastogenesis and in mature osteoclasts. The findings strengthen the relation between osteoclasts and the mononuclear phagocyte system. Furthermore, they suggest that M-CSF acts directly on osteoclast precursors and on mature osteoclasts during osteoclastogenesis.
Myristoylation of the G alpha i2 polypeptide, a G protein alpha subunit, is required for its signaling and transformation functions.
Authors: Gallego C , Gupta S K , Winitz S , Eisfelder B J , Johnson G L
Published: May 2006

GTPase-inhibiting mutations of the alpha subunit (alpha i2) of the G protein, Gi2, result in constitutive activation of alpha i2 signal transduction functions. GTPase-inhibited alpha i2 mutant polypeptides, referred to as gip2 oncoproteins, have glutamine-205 mutated to leucine (alpha i2Q205L). Expression of the alpha i2Q205L polypeptide inhibits adenylyl cyclase stimulation, constitutively activates p42 mitogen-activated protein kinase, and transforms Rat 1a fibroblasts. The alpha i2 polypeptides are N-terminal-myristoylated, but the function of myristoylation is unclear in alpha i2 signal transduction. We have tested the requirement for myristoylation on the ability of the alpha i2Q205L mutant polypeptide to constitutively regulate signal pathways and cell transformation. When expressed in Rat 1a cells, the nonmyristoylated alpha i2Q205L polypeptide is membrane associated but is unable to regulate adenylyl cyclase or p42 mitogen-activated protein kinase and does not induce cellular transformation. We conclude that myristoylation is absolutely necessary for alpha i2Q205L signal transduction and regulation of effector enzymes in the cell.
Selective expression of human X chromosome-linked green opsin genes.
Authors: Winderickx J , Battisti L , Motulsky A G , Deeb S S
Published: May 2006

The human red and green photopigments are specified by genes on the long arm of the X chromosome (Xq28). In individuals with normal color vision, the locus was proposed to consist of a single red pigment gene upstream of one or more copies of green pigment genes. The presence of a single red pigment gene in the array was confirmed by demonstration of only one retinal mRNA transcript coding for the red opsin. In individuals with multiple green pigment genes, it is unknown whether all genes are expressed. We analyzed the sequence of red- and green-specific mRNA from retinas of individuals with multiple green pigment genes in comparison with the corresponding genomic DNA sequences. The data showed that only a single green pigment gene is expressed. We therefore suggest that a locus control-like element, already known to be located 3.8 kilobases upstream of the transcription initiation site of the red pigment gene, allows transcription of only a single copy of the green pigment genes, probably the most proximal copy. This finding provides an explanation for the not-infrequent presence of 5' green-red hybrid genes in individuals with normal color vision. Such hybrid genes are usually associated with defective color vision. We suggest that 5' green-red hybrid genes produce defective color vision only when their position in the gene array allows expression in the retinal cone cells.
Roles of selection and recombination in the evolution of type I restriction-modification systems in enterobacteria.
Authors: Sharp P M , Kelleher J E , Daniel A S , Cowan G M , Murray N E
Published: May 2006

Restriction-modification systems can protect bacteria against viral infection. Sequences of the hsdM gene, encoding one of the three subunits of type I restriction-modification systems, have been determined for four strains of enterobacteria. Comparison with the known sequences of EcoK and EcoR124 indicates that all are homologous, though they fall into three families (exemplified by EcoK, EcoA, and EcoR124), the first two of which are apparently allelic. The extent of amino acid sequence identity between EcoK and EcoA is so low that the genes encoding them might be better termed pseudoalleles; this almost certainly reflects genetic exchange among highly divergent species. Within the EcoK family the ratio of intra- to interspecific divergence is very high. The extent of divergence between the genes from Escherichia coli K-12 and Salmonella typhimurium LT2 is similar to that for other genes with the same level of codon usage bias. In contrast, intraspecific divergence (between E. coli strains B and K-12) is extremely high and may reflect the action of frequency-dependent selection mediated by bacteriophages. There is also evidence of lateral transfer of a short sequence between E. coli and S. typhimurium.
Photosynthetic membrane topography: quantitative in situ localization of photosystems I and II.
Authors: Mustardy L , Cunningham F X , Gantt E
Published: May 2006

An immunolabeling approach was developed for quantitative in situ labeling of photosystems I and II (PSI and PSII). Photosynthetic membranes from the phycobilisome-containing red alga Porphyridium cruentum were isolated from cells in which different photosystem compositions were predetermined by growing cells in green light (GL) or red light (RL). Based on phycobilisome densities per membrane area of 390 per m2 (GL) and 450 per m2 (RL) and the PSI reaction center (P700) and PSII reaction center (QA) content, the photosystem densities per m2 of membrane were calculated to be 2520 PSI in GL and 1580 in RL and 630 PSII in GL and 1890 in RL. PSI was detected in the membranes with 10-nm Au particles conjugated to affinity-purified anti-PSI, and PSII was detected with 15-nm Au particles conjugated to anti-PSII. Distribution of Au particles appeared relatively uniform, and the degree of labeling was consistent with the calculated photosystem densities. However, the absolute numbers of Au-labeled sites were lower than would be obtained if all reaction center monomers were labeled. Specific labeling of PSI was 25% in GL and RL membranes, and PSII labeling was 33% in GL but only 17% in RL membranes. An IgG-Au particle is larger than a monomer of either photosystem and could shield several closely packed photosystems. We suggest that clustering of photosystems exists and that the cluster size of PSI is the same in GL and RL cells, but the PSII cluster size is 2 times greater in RL than in GL cells. Such variations may reflect changes in functional domains whereby increased clustering can maximize the cooperativity between the photosystems, resulting in enhancement of the quantum yield.
A binary plasmid system for shuffling combinatorial antibody libraries.
Authors: Collet T A , Roben P , O'Kennedy R , Barbas C F , Burton D R , Lerner R A
Published: May 2006

We have used a binary system of replicon-compatible plasmids to test the potential for promiscuous recombination of heavy and light chains within sets of human Fab fragments isolated from combinatorial antibody libraries. Antibody molecules showed a surprising amount of promiscuity in that a particular heavy chain could recombine with multiple light chains with retention of binding to a protein antigen. The degree to which a given heavy chain productively paired with any light chain to bind antigen varied from 43% to 100% and depended strongly on the heavy-chain sequence. Such productive crosses resulted in a set of Fab fragments of similar apparent binding constants, which seemed to differ mainly in the amount of active Fab fragment produced in the bacterial cell. The dominance of the heavy chain in the antibody-antigen interaction was further explored in a set of directed crosses, in which heavy and light chains derived from antigen-specific clones were crossed with nonrelated heavy and light chains. In these crosses, an Fab fragment retained antigen binding only if it contained a heavy chain from an antigen-specific clone. In no case did the light chain confer detectable affinity when paired with indifferent heavy chains. The surprising promiscuity of heavy chains has ramifications for the evaluation of the diversity of combinatorial libraries made against protein antigens and should allow the combination of one such promiscuous heavy chain with an engineered light chain to form an Fab fragment carrying synthetic cofactors to assist in antibody catalysis.
Structural analysis of ternary complexes of vaccinia RNA polymerase.
Authors: Hagler J , Shuman S
Published: May 2006

The structure of the elongation complex of vaccinia RNA polymerase halted at discrete template positions was examined by DNase I footprinting. The leading edge of the footprint bore a constant relationship to the catalytic template position, being 22-24 nucleotides (nt) in advance on the nontemplate strand and 17 nt on the template strand. DNase hypersensitivity of the nontemplate strand at the leading edge suggested that the DNA might be distorted as it entered the polymerase molecule. The region of DNA unwinding at the transcription bubble extended at least 12 nt 5' from the catalytic center, as indicated by the reactivity of adenosine residues to diethylpyrocarbonate. Cu-phenanthroline-hypersensitive sites located 13 nt 5' and 4 nt 3' of the growing point appeared to demarcate the margins of the bubble. Strand asymmetry of chemical modification within the bubble was consistent with an RNA-DNA hybrid of no more than 10 base pairs.
Relationship of human immunodeficiency virus type 1 sequence heterogeneity to stage of disease.
Authors: McNearney T , Hornickova Z , Markham R , Birdwell A , Arens M , Saah A , Ratner L
Published: May 2006

V3 envelope sequences were determined from amplified human immunodeficiency virus type 1 (HIV-1) sequences of uncultivated leukocytes obtained sequentially from four infected adults over the course of infection. Lower levels of sequence heterogeneity were noted in samples obtained early in HIV-1 infection, prior to CD4 depletion, than in samples obtained at later times during disease. The pattern of amino acid sequence divergence included nonrandom changes, with evidence of sequence variants arising from HIV-1 quasi-species present earlier in infection. Consensus sequences for isolates obtained early after infection from different patients demonstrated a high level of conservation with one another and a consensus sequence for macrophage-tropic HIV-1 isolates. These findings suggest that a highly restricted subset of HIV-1 quasi-species can be transmitted and can establish infection.
Adaptive cellular interactions in the immune system: the tunable activation threshold and the significance of subthreshold responses.
Authors: Grossman Z , Paul W E
Published: May 2006

A major challenge for immunologists is to explain how the immune system adjusts its responses to the microenvironmental context in which antigens are recognized. We propose that lymphocytes achieve this by tuning and updating their responsiveness to recurrent signals. In particular, cellular anergy in vivo is a dynamic state in which the threshold for a stereotypic mode of activation has been elevated. Anergy is associated with other forms of cellular activity, not paralysis. Cells engaged in such subthreshold interactions mediate functions such as maintenance of immunological memory and control of infections. In such interactions, patterns of signals are recognized and classified and evoke selective responses. The robust mechanism proposed for segregation of suprathreshold and subthreshold immune responses allows lymphocytes to use recognition of self-antigens in executing physiological functions. Autoreactivity is allowed where it is dissociated from uncontrolled aggression.
Differential regulation of neuropeptide mRNA expression in intrastriatal striatal transplants by host dopaminergic afferents.
Authors: Campbell K , Wictorin K , Björklund A
Published: May 2006

The effects of dopamine-specific manipulations on neuropeptide gene expression in intrastriatal grafts of fetal striatal tissue were studied by quantitative in situ hybridization histochemistry, using 35S-labeled oligonucleotide probes. Messenger RNA transcripts for the striatal neuropeptides preproenkephalin (PPE) and preprotachykinin (PPT) were detected in neurons forming discrete patches in the striatal grafts. The relative abundance of PPE and PPT mRNA-expressing neurons within the graft patches (51-54%) was similar to that found in normal caudate-putamen. In specimens with intact dopamine afferents the expression of PPE mRNA in grafted neurons was similar to that found in normal caudate putamen, whereas the hybridization signal for PPT mRNA was 27% higher in the graft neurons than in the normal caudate-putamen. Removal of host dopaminergic afferents by 6-hydroxydopamine lesions of the ipsilateral mesostriatal dopamine pathway increased the hybridization signal for PPE mRNA both in the grafts (+84%) and in the spared ipsilateral host caudate-putamen (+125%), whereas the PPT signal was reduced by 53% in the grafts and by 51% in the remaining host caudate-putamen. Similarly, chronic treatment of grafted animals with the dopamine receptor antagonist haloperidol (2 mg/kg per day for 10 days) produced a 146% increase in the PPE signal in the grafts and a 175% increase in the intact contralateral caudate-putamen, whereas the signal for PPT mRNA was again decreased by 52% and 51% in the grafts and host caudate-putamen, respectively. These results show that the host nigrostriatal dopamine pathway differentially regulates enkephalin and substance P gene expression within striatal grafts and thereby exerts a tonic functional influence over grafted striatal neurons.
Identification of a mouse brain cDNA that encodes a protein related to the Alzheimer disease-associated amyloid beta protein precursor.
Authors: Wasco W , Bupp K , Magendantz M , Gusella J F , Tanzi R E , Solomon F
Published: May 2006

We have isolated a cDNA from a mouse brain library that encodes a protein whose predicted amino acid sequence is 42% identical and 64% similar to that of the amyloid beta protein precursor (APP). This 653-amino acid protein, which we have termed the amyloid precursor-like protein (APLP), appears to be similar to APP in overall structure as well as amino acid sequence. The amino acid homologies are concentrated within three distinct regions of the two proteins where the identities are 47%, 54%, and 56%. The APLP cDNA hybridizes to two messages of approximately 2.4 and 1.6 kilobases that are present in mouse brain and neuroblastoma cells. Polyclonal antibodies raised against a peptide derived from the C terminus of APLP stain the cytoplasm in a pattern reminiscent of Golgi staining. In addition to APP, APLP also displays significant homology to the Drosophila APP-like protein APPL and a rat testes APP-like protein. These data indicate that the APP gene is a member of a strongly conserved gene family. Studies aimed at determining the functions of the proteins encoded by this gene family should provide valuable clues to their potential role in Alzheimer disease neuropathology.
Retinal rods and cones have distinct G protein beta and gamma subunits.
Authors: Peng Y W , Robishaw J D , Levine M A , Yau K W
Published: May 2006

Guanine nucleotide-binding proteins (G proteins) involved in transmembrane signal-transduction processes are heterotrimers composed of alpha, beta, and gamma subunits. The alpha subunit shows great diversity and is thought to confer functional specificity to a particular G protein. By contrast, the beta and gamma subunits appear much less diverse; in particular, the beta subunit is believed to have no role in G protein specificity. Using immunocytochemistry, we found distinct distribution patterns for different beta and gamma subunits in the retina. In particular, rod and cone photoreceptors, which both subserve phototransduction but differ in light-response properties, have different beta and gamma subunits in their outer segments. Thus, the G protein mediating phototransduction shows cell-specific forms of the beta and gamma subunits in addition to the alpha subunit. This surprising finding supports the hypothesis that these subunits may also contribute to functional specificity of a G protein.
Molecular cloning and primary structure of the human blood group RhD polypeptide.
Authors: Le van Kim C , Mouro I , Chérif-Zahar B , Raynal V , Cherrier C , Cartron J P , Colin Y
Published: May 2006

The RH (rhesus) blood group locus from RhD-positive donors is composed of two homologous structural genes, one of which encodes the Cc and Ee polypeptides, whereas the other, which is missing in the RhD-negative condition, encodes the D protein that carries the major antigen of the RH system. Recently, different splicing isoforms transcribed from the CcEe gene were isolated. We report now the characterization of two other Rh clones, RhII and RhXIII, generated by alternative choices for poly(A) addition sites that were identified as the RhD gene transcripts. That these cDNAs represented the RhD messenger and that the previously described Rh clones were derived from the CcEe gene was demonstrated by amplification of RhII/XIII sequences only from D-positive genomes and by cloning and sequencing of D- and CcEe-specific gene fragments. The predicted translation product of the RhD mRNA is a 417-amino acid protein (M(r) = 45,500) that exhibited a similar membrane organization with 13 bilayer-spanning domains compared with the polypeptide encoded by the CcEe gene. The D and Cc/Ee polypeptides differ by 36 amino acid substitutions (8.4% divergence), but the NH2- and COOH-terminal regions of the two proteins are well conserved. Similarly, five of the six cysteine residues of the Cc/Ee proteins were conserved in the D protein, including the unique exofacial cysteine, which is critical for antigenic reactivity. The sequence homology between the Cc/Ee and D proteins supports the concept that the genes encoding these polypeptides have evolved by duplication of a common ancestor gene.
Uranium-loaded apoferritin with antibodies attached: molecular design for uranium neutron-capture therapy.
Authors: Hainfeld J F
Published: May 2006

A method is described to deliver 235U to tumors; the isotope would then be fissioned by incident neutrons, producing localized lethal radiation sufficient for therapy. Apoferritin was loaded with an average of approximately 800 238U atoms per molecule. Stability of the loaded apoferritin in solution was improved, so that only 8% loss of uranium occurred after 8 days at pH 7. Fab' antibody fragments were covalently attached to the uranium-loaded apoferritin, and the immunoreactivity of the conjugate was 92% of that for antibody alone. Such bio-uranium constructions should provide significant advantages over boronated antibodies to meet the requirements for clinical neutron-capture therapy.
Cytoskeletal integrity in interphase cells requires protein phosphatase activity.
Authors: Eriksson J E , Brautigan D L , Vallee R , Olmsted J , Fujiki H , Goldman R D
Published: May 2006

Phosphorylation by protein kinases has been established as a key factor in the regulation of cytoskeletal structure. However, little is known about the role of protein phosphatases in cytoskeletal regulation. To assess the possible functions of protein phosphatases in this respect, we studied the effects of the phosphatase inhibitors calyculin A, okadaic acid, and dinophysistoxin 1 (35-methylokadaic acid) on BHK-21 fibroblasts. Within minutes of incubation with these inhibitors, changes are seen in the structural organization of intermediate filaments, followed by a loss of microtubules, as assayed by immunofluorescence. These changes in cytoskeletal structure are accompanied by a rapid and selective increase in vimentin phosphorylation on interphase-specific sites, and they are fully reversible after removal of calyculin A. The results indicate that there is a rapid phosphate turnover on cytoskeletal intermediate filaments and further suggest that protein phosphatases are essential for the maintenance and structural integrity of two major cytoskeletal components.
Molecular cloning and functional expression of a brain-specific somatostatin receptor.
Authors: Bruno J F , Xu Y , Song J , Berelowitz M
Published: May 2006

The PCR and conventional library screening were used to clone the brain-specific somatostatin receptor rSSTR-4 from a rat genomic library. The deduced amino acid sequence encodes a protein of 384 amino acids and displays structural and sequence homologies with members of the G protein-receptor superfamily. The amino acid sequence of rSSTR-4 is 60% and 48% identical to that of somatostatin receptors SSTR-1 and SSTR-2, respectively, two recently cloned subtypes. Competition curve analysis of the binding properties of the receptor transiently expressed in COS-1 cells revealed a higher apparent affinity for somatostatin 14 than for somatostatin 28. In contrast, the somatostatin analogs SMS 201-995, IM 4-28, and MK-678 failed to displace specific binding in transfected cells. These characteristics resemble the pharmacological binding properties of the previously described brain-specific somatostatin-receptor subtype. Examination of the tissue distribution of mRNA for rSSTR-4 revealed expression limited to various brain regions with highest levels in the cortex and hippocampus. Thus, based on the pharmacology and tissue localization of this receptor, we conclude that rSSTR-4 represents a brain-specific somatostatin receptor.
Specific inhibition of human immunodeficiency virus type 1 replication by antisense oligonucleotides: an in vitro model for treatment.
Authors: Lisziewicz J , Sun D , Klotman M , Agrawal S , Zamecnik P , Gallo R
Published: May 2006

We have developed a culture system, simulating in vivo conditions of human immunodeficiency virus type 1 (HIV-1) infection, to evaluate the long-term efficacy of antisense oligonucleotide treatment. Five oligonucleotide phosphorothioates (28-mers), complementary to different regions of HIV-1 RNA, blocked replication of the virus in a sequence-specific manner at 1 microM concentration. Variations in antiviral activity were seen among the different oligonucleotides, revealing an effect of target selection. Mismatched or random oligonucleotide phosphorothioates delayed, but did not completely inhibit, HIV-1 replication. In the case of inhibition by a splice-acceptor-site antisense oligodeoxynucleotide, a break-through phenomenon occurred after 25 days of treatment, suggesting the development of an "escape mutant." This result did not occur when the inhibitory oligodeoxynucleotides were complementary to the primary-sequence areas of the rev-responsive element and rev-1 genes. Sequential treatment of HIV-1-infected cells with a combination of different antisense oligonucleotides, each administered once, also prevented the development of escape mutants. Our results suggest that chemotherapy based on specifically targeted antisense-oligonucleotide phosphorothioates may be an effective method for reducing the viral burden in HIV-1-infected individuals at clinically achievable oligonucleotide concentrations.
NAD+ and NADH regulate an ATP-dependent kinase that phosphorylates enzyme I of the Escherichia coli phosphotransferase system.
Authors: Dannelly H K , Roseman S
Published: May 2006

Crude extracts of Escherichia coli contain a protein kinase, EI-K, that phosphorylates enzyme I (EI) of the phosphoenolpyruvate:glycose phosphotransferase system (PTS). Phosphorylation occurs at the active site histidine residue. The activity of EI-K was lost during purification. However, kinase activity was restored by adding NAD+ or NADP+.NADH reversed NAD+ activation of the kinase, and the level of EI-K activity was dependent on the NAD+/NADH ratio. Although crude preparations of EI-K showed no NAD+ requirement, they were completely inhibited by NADH, either in the assay mixture or when the enzyme was pretreated and the NADH was removed prior to the assay. NAD+ restored full activity to the NADH-pretreated inactive fractions. The results suggest that EI-K contains a bound cofactor that is lost during purification and that may be analogous to NAD+. EI-K activity may serve to link some of the diverse functions of the PTS, such as sugar transport, to the metabolic state of the cell.
Genetic control of murine hematopoietic stem cell pool sizes and cycling kinetics.
Authors: Phillips R L , Reinhart A J , Van Zant G
Published: May 2006

Bone marrow from each of two inbred mouse strains, C57BL/6J and DBA/2J, was highly enriched for stem cells using flow cytometry and was divided into two stem cell subpopulations using the mitochondrial dye rhodamine 123 (Rh-123). The Rh-123lo population was determined to be more primitive than Rh-123hi based on the expression of stem cell markers such as the c-kit protooncogene (stem cell factor receptor) and the Ly-6A/E stem cell antigen (Sca-1) as well as the lack of in vitro colony-forming ability. Compared to DBA/2J mice, marrow from the C57BL/6J strain consistently showed a higher proportion of "very primitive" (Rh-123lo) cells, suggesting that the sizes of functionally distinct stem cell subpopulations are maintained under precise genetic control. Marrow from both strains exposed to the cytotoxic drug 5-fluorouracil showed a dramatic increase in the proportion of Rh-123lo cells within 2 days as repopulation began. Marrow subpopulations returned to pretreatment proportions by the eighth day in DBA/2J mice but not until 14 days in C57BL/6J mice. This intrinsic difference in 5-fluorouracil recovery time was attributed to an increase rate of stem cell cycling in DBA/2J relative to C57BL/6J mice. When stem cell factor was injected into a C57BL/6J<-->DBA/2J allophenic mouse, blood cell chimerism shifted markedly but transiently toward the DBA/2J genotype, suggesting that the DBA/2J target population, because of an inherent kinetic advantage, was able to respond faster to the cytokine. A model is proposed that is based on these and our earlier observations to explain this strain-specific stem cell behavior and offer new insights into the genetic control of stem cell cycling and population dynamics.
Suppression of the onset of myelination extends the permissive period for the functional repair of embryonic spinal cord.
Authors: Keirstead H S , Hasan S J , Muir G D , Steeves J D
Published: May 2006

In an embryonic chicken, transection of the thoracic spinal cord prior to embryonic day (E) 13 (of the 21-day developmental period) results in complete neuroanatomical repair and functional locomotor recovery. Conversely, repair rapidly diminishes following a transection on E13-E14 and is nonexistent after an E15 transection. The myelination of fiber tracts within the spinal cord also begins on E13, coincident with the transition from permissive to restrictive repair periods. The onset of myelination can be delayed (dysmyelination) until later in development by the direct injection into the thoracic cord on E9-E12 of a monoclonal antibody to galactocerebroside, plus homologous complement. In such a dysmyelinated embryo, a subsequent transection of the thoracic cord as late as E15 resulted in complete neuroanatomical repair and functional recovery (i.e., extended the permissive period for repair).
Carbonic anhydrase in the membrane of the endoplasmic reticulum of male rat liver.
Authors: Ono Y , Ridderstråle Y , Forster R E , Chu Z G , Dodgson S J
Published: May 2006

We have prepared subcellular fractions of male rat liver homogenate by the method of Lewis and Tata [Lewis, J. A. & Tata, J. R. (1973) J. Cell Sci. 23, 447-459], further purifying the membranes of the microsomal fraction by exposure to 0.01% Triton X-100 and centrifugation. We determined the purity of the fractions with marker enzymes and measured carbonic anhydrase (CA; EC 4.2.1.1) activity in intact and solubilized particulates with 18O exchange between CO2/HCO3- and water. We measured the concentration of CA by titration with a sulfonamide inhibitor, ethoxzolamide, obtaining an average value of 3.8 mumol/mg of microsomal membrane protein. The equilibrium constant for binding ethoxzolamide was 0.49 x 10(-9) M. The Km for CO2 was 1.7 mM and the turnover number was 560,000 sec-1, characterizing this as a membrane-bound, high-activity isozyme of type IV. By electron microscopy of tissue sections after staining with a cobalt precipitation technique, CA was seen in small cytoplasmic vesicles in hepatocytes and in microsomal particles and membranes. There was a sulfonamide-resistant (isozyme type III) and a sulfonamide-sensitive (isozyme type II) CA in the cytosol but none in the rapidly sedimenting endoplasmic reticulum. We conclude that there is no CA normally within the matrix of the cell endoplasmic reticulum but that the CA type III found in the microsome may have been captured from the cytosol during resealing. Thus the adult male rat hepatocyte contains CA type IV in the membrane of the endoplasmic reticulum and CA type II and CA type III in the cytoplasm.
Molecular mimicry of hepatitis B surface antigen by an anti-idiotype-derived synthetic peptide.
Authors: Pride M W , Shi H , Anchin J M , Linthicum D S , LoVerde P T , Thakur A , Thanavala Y
Published: May 2006

Monoclonal antibody 2F10 is an "internal-image" anti-idiotype (anti-id) antibody capable of mimicking the group-specific "a" determinant of human hepatitis B surface antigen (HBsAg). By mRNA sequencing and computer-assisted molecular modeling of monoclonal antibody 2F10, we identified a 15-amino acid region of the heavy-chain hypervariable region that has partial residue homology with sequences of the "a" determinant epitopes of HBsAg. We have established that a linear 15-mer peptide from a contiguous region on the anti-id antibody can (i) generate anti-HBsAg-specific antibodies when injected into mice, (ii) prime murine lymph node cells for in vitro HBsAg-specific T-cell proliferative responses, and (iii) stimulate in vitro human CD4+ T cells that were primed in vivo to HBsAg by natural infection with hepatitis B virus or vaccination with a commercially available HBsAg vaccine. Significantly, this peptide could also stimulate CD4+ T cells of human hepatitis B virus carriers. We conclude that a 15-mer peptide derived from the anti-id sequence can duplicate the B- and T-cell stimulatory activity of the intact anti-id antibody and the antigen that is mimicked, HBsAg.
Patterns of spatiotemporal organization in an "ambiquitous" enzyme model.
Authors: Marmillot P , Hervagault J F , Welch G R
Published: May 2006

Many enzymes in pathways such as glycolysis associate reversibly with cellular substructures. The spatiotemporal behavior of a "limit-cycle" oscillation model is studied under the condition that the "ambiquitous" oscillophor, phosphofructokinase, is partitioned between "bulk-phase" and "bound" forms in a heterogeneous system. Computer simulation demonstrates the occurrence of sustained, wave-like spatiotemporal patterns of chemical concentration in the bulk medium. Kinetic dissimilarity among the localized populations of bound enzyme leads to a "polarity" effect in the wave phenomenon. It is suggested that a key physiological role of the limit-cycle regime is to engender a rapid, site-to-site, signal-transmission modality in large eukaryotic (e.g., mammalian) cells.
Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic.
Authors: Kirnbauer R , Booy F , Cheng N , Lowy D R , Schiller J T
Published: May 2006

Infection by certain human papillomavirus types is regarded as the major risk factor in the development of cervical cancer, one of the most common cancers of women worldwide. Analysis of the immunogenic and structural features of papillomavirus virions has been hampered by the inability to efficiently propagate the viruses in cultured cells. For instance, it has not been established whether the major capsid protein L1 alone is sufficient for virus particle assembly. In addition, it is not known whether L1, L2 (the minor capsid protein), or both present the immunodominant epitopes required for induction of high-titer neutralizing antibodies. We have expressed the L1 major capsid proteins of bovine papillomavirus type 1 and human papillomavirus type 16 in insect cells via a baculovirus vector and analyzed their conformation and immunogenicity. The L1 proteins were expressed at high levels and assembled into structures that closely resembled papillomavirus virions. The self-assembled bovine papillomavirus L1, in contrast to L1 extracted from recombinant bacteria or denatured virions, also mimicked intact bovine papillomavirus virions in being able to induce high-titer neutralizing rabbit antisera. These results indicate that L1 protein has the intrinsic capacity to assemble into empty capsid-like structures whose immunogenicity is similar to infectious virions. This type of L1 preparation might be considered as a candidate for a serological test to measure antibodies to conformational virion epitopes and for a vaccine to prevent papillomavirus infection.
Ha-ras oncogene activation in mammary glands of N-methyl-N-nitrosourea-treated rats genetically resistant to mammary adenocarcinogenesis.
Authors: Lu S J , Archer M C
Published: May 2006

A single dose of N-methyl-N-nitrosourea given to sexually immature female Buf/N rats produces a high incidence of mammary adenocarcinomas. A large percentage of these tumors contain the Ha-ras oncogene, activated by a G----A transition at the second nucleotide of codon 12. Copenhagen rats, on the other hand, are completely resistant to mammary tumor induction by a number of carcinogens, including N-methyl-N-nitrosourea. Here we show, using a sensitive method involving PCR, that codon 12 Ha-ras mutations occur in the mammary glands of both Buf/N and Copenhagen rats 30 days after N-methyl-N-nitrosourea treatment. These mutations were evenly distributed among individual mammary glands and were present in purified mammary epithelial cells. In Buf/N rats, the fraction of cells containing a mutated Ha-ras allele increased by a factor of 10-100 between 30 and 60 days, whereas in Copenhagen rats, there was no such increase during this time period. We conclude that the resistance of the Copenhagen rat to mammary carcinogenesis is not due to a defect in initiation but rather appears to be due to the inability of cells containing a mutated ras allele to undergo sustained clonal expansion.
Nonconservative segregation of parental nucleosomes during simian virus 40 chromosome replication in vitro.
Authors: Sugasawa K , Ishimi Y , Eki T , Hurwitz J , Kikuchi A , Hanaoka F
Published: May 2006

Simian virus 40 chromosomes can be replicated in vitro with the same set of purified proteins required for the replication of naked DNA containing the viral origin. With these reconstituted systems, the fate of parental histones during replication was examined in vitro. The assembly of nucleosomes on replicating chromosomes was hardly affected by the presence of simultaneously replicating naked DNA competitor, suggesting that replication forks can traverse nucleosomes without the displacement of histones. Moreover, we demonstrate that the nascent nucleosomes were distributed almost equally between the leading and lagging strands. This distributive mode of nucleosome segregation favors the propagation of parental chromatin structures to both daughter cells, which can maintain cellular functions dictated by these structures during cell proliferation.
Isolation of a src homology 2-containing tyrosine phosphatase.
Authors: Plutzky J , Neel B G , Rosenberg R D
Published: May 2006

Tyrosine phosphorylation is controlled by the opposing actions of tyrosine kinases and phosphotyrosine phosphatases (PTPs). src homology 2 domains (SH2) are found in several types of signaling proteins, including some tyrosine kinases. These domains bind phosphotyrosyl proteins and thus help promote signal transduction. Using mixed oligonucleotide-directed polymerase chain reactions, two previously undescribed rat PTP cDNA fragments were generated. Through subsequent screening of rat megakaryocyte and human erythroleukemia libraries, we obtained a full-length coding sequence for one of these fragments. This cDNA, SH-PTP1, encodes a tyrosine phosphatase containing two highly conserved SH2 domains. SH-PTP1, with a 2.4-kilobase mRNA, a predicted open reading frame of 595 amino acids, and a structure suggesting a nontransmembrane protein, is expressed primarily in hematopoietic and epithelial cells. When expressed in Escherichia coli, SH-PTP1 possesses PTP activity. The structure of SH-PTP1 establishes an additional branch of the tyrosine phosphatase family and suggests mechanisms through which tyrosine phosphatases might participate in signal transduction pathways.
Education for industrial ecology.
Authors: Starr C
Published: May 2006

The societal issues relevant to the subject of industrial ecology call for the participation of broadly educated engineers who can integrate their technology with the social, political, environmental, and economic aspects of its applications. This increase in the responsibility of traditional engineering to include the end-use, obsolescence, and disposition of technical products signals a new era for engineering education.
Function of specific 2'-hydroxyl groups of guanosines in a hammerhead ribozyme probed by 2' modifications.
Authors: Williams D M , Pieken W A , Eckstein F
Published: May 2006

The importance of the 2'-hydroxyl group of several guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. Five ribozymes in which single guanosine residues were substituted with 2'-amino-, 2'-fluoro-, or 2'-deoxyguanosine were chemically synthesized. The comparison of the catalytic activity of the three 2' modifications at a specific position allows conclusions about the functional role of the parent 2'-hydroxyl group. Substitutions of nonconserved nucleotides within the ribozyme caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, when either of the guanosines within the single-stranded loop between stem I and stem II of the ribozyme was replaced by 2'-deoxyguanosine or 2'-fluoro-2'-deoxyguanosine, the catalytic activities of the resulting ribozymes were reduced by factors of at least 150. The catalytic activities of the corresponding ribozymes containing 2'-amino-2'-deoxyguanosine substitutions at these positions, however, were both reduced by factors of 15. These effects resulted from decreases in the respective kcat values, whereas variations in the Km values were comparatively small. A different pattern of reactivity of the three 2' modifications was observed at the guanosine immediately 3' to stem II of the ribozyme. Whereas both 2'-deoxyguanosine and 2'-amino-2'-deoxyguanosine at this position showed catalytic activity similar to that of the unmodified ribozyme, the activity of the corresponding 2'-fluoro-2'-deoxyguanosine-containing ribozyme was reduced by a factor of 15. The implications of these substitution-specific reactivities on the functional role of the native 2'-hydroxyl groups are discussed.
Preservation of metabolic activity in lyophilized human erythrocytes.
Authors: Goodrich R P , Sowemimo-Coker S O , Zerez C R , Tanaka K R
Published: May 2006

Normal human erythrocytes (RBC) were freeze-dried under conditions that caused minimal modification in normal RBC metabolic activities. Because of the known effects of long-term storage on metabolic activities, we studied the effects of our lyophilization process on RBC metabolism. Of all the metabolic enzymes studied, only triosephosphate isomerase (D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1), enolase (2-phospho-D-glyceratehydro-lyase, EC 4.2.1.11), and pyruvate kinase (ATP:pyruvate O2-phosphotransferase, EC 2.7.1.40) were decreased when compared with fresh control nonlyophilized RBC. The activities of these enzymes did not differ significantly from those of blood bank RBC. Concentrations of high-energy intermediates, ATP, and 2,3-diphosphoglycerate, along with lactate and ATP production were decreased in lyophilized RBC. No enzymes of the pentose phosphate shunt were altered during lyophilization. In addition, our data show that lyophilized RBC possess an intact capacity to (i) synthesize adenine nucleotides and (ii) reduce MetHb to Hb and, thus, maintain the Hb in a functional physiologic state similar to fresh nonlyophilized RBC. The present study demonstrates the possibility of lyophilizing RBC in a manner that maintains normal metabolic and enzymatic function upon rehydration.
Structure of clonal and polyclonal cell arrays in chimeric mouse retina.
Authors: Williams R W , Goldowitz D
Published: May 2006

One of the most striking results of recent cell-lineage studies of vertebrate retina is the marked variability in the size and types of clones marked by retroviral transfection and dye injection of embryonic progenitor cells. Is this variability due to microenvironmental modulation of cell determination, to lineage restriction, or to experimental perturbation of the progenitor cells? We have taken advantage of species-specific DNA probes to mark groups of lineage-related cells in experimental mouse chimeras. This method of marking cells has two distinct advantages over previous methods: direct manipulation of progenitor cells is avoided, and clones are established at an earlier stage of retinal development. The most notable feature of retinal cohorts in chimeras is their structural uniformity--each is a solid radial array that contains the same ratio of major cell types as the retina itself. This is true even of the smallest monoclonal cohorts, which contain fewer than 200 cells. Our results provides compelling empirical support for the hypothesis that the murine retina is made up of hundreds of relatively homogeneous radial units, each derived from single retinal precursor cells. This finding is inconsistent with micro-environmental modulation of clone structure early in development. We raise the possibility that the heterogeneity among clones marked by dye injection and transfection is due to progressive lineage restriction or to experimental perturbation of the retinal progenitor cells.