bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutational and in vivo methylation analysis of F-factor PifC protein binding to the pif operator and the region containing the primary origin of mini-F replication.
Authors: Miller J F , Malamy M H
Published: May 2006

We have used in vivo methods to identify multiple DNA-binding sites for the negatively autoregulated mini-F replication factor PifC. Sequence analysis of pif operator constitutive mutants, isolated as insensitive to repression by PifC, establishes the structure of pifO. This site contains a 17-base-pair (bp) region of dyad symmetry with 7-bp perfect inverted repeats separated by 3 bp. In vivo DNA methylation studies with dimethyl sulfate show that the reactivity of five of six guanine residues in the pifO region is altered in the presence of PifC protein. In addition, there are several sites of PifC-dependent methylation enhancement and protection upstream of pifO within repeated sequences bearing homology to pifO. The significance of the repeated PifC binding sequences and their relationship to the primary origin of mini-F replication (oriV1) are discussed.
Site-saturation studies of beta-lactamase: production and characterization of mutant beta-lactamases with all possible amino acid substitutions at residue 71.
Authors: Schultz S C , Richards J H
Published: May 2006

A mutagenic technique that "saturates" a particular site in a protein with all possible amino acid substitutions was used to study the role of residue 71 in beta-lactamase (EC 3.5.2.6). Threonine is conserved at residue 71 in all class A beta-lactamases and is adjacent to the active site Ser-70. All 19 mutants of the enzyme were characterized by the penam and cephem antibiotic resistance they provided to Escherichia coli LS1 cells. Surprisingly, cells producing any of 14 of the mutant beta-lactamases displayed appreciable resistance to ampicillin; only cells with mutants having Tyr, Trp, Asp, Lys, or Arg at residue 71 had no observable resistance to ampicillin. However, the mutants are less stable to cellular proteases than wild-type enzyme is. These results suggest that Thr-71 is not essential for binding or catalysis but is important for stability of the beta-lactamase protein. An apparent change in specificity indicates that residue 71 influences the region of the protein that accommodates the side chain attached to the beta-lactam ring of the substrate.
Mechanism by which contact with plant cuticle triggers cutinase gene expression in the spores of
Fusarium solani
f. sp.
pisi
Authors: Woloshuk Charles P. , Kolattukudy P. E.
Published: May 2006

Spores of the phytopathogenic fungus
Fusarium solani
f. sp.
pisi
were shown to produce the extracellular enzyme, cutinase, only when cutin or cutin hydrolysate was added to the spore suspension. Dihydroxy-C
16
acid and trihydroxy-C
18
acid, which are unique cutin monomers, showed the greatest cutinase-inducing activity. Experiments with several compounds structurally related to these fatty acids suggested that both a ω-hydroxyl and a midchain hydroxyl are required for cutinase-inducing activity. Cutinase appeared in the medium 30-45 min after the addition of the inducers to the spore suspension, and the activity level increased for 6 hr. Addition of cycloheximide (5 μg/ml) completely inhibited cutinase production, suggesting that protein synthesis was involved in the increase of cutinase activity. Immunoblot analysis with rabbit antibodies prepared against cutinase showed that cutinase protein increased in parallel with the increase in enzyme activity. Measurement of cutinase-specific RNA levels by dot-blot hybridization with
32
P-labeled cutinase cDNA showed that the cutinase gene transcripts could be detected within 15 min after addition of the inducers. Addition of exogenous cutinase greatly enhanced the level of cutinase gene transcripts induced by cutin. These results strongly suggest that the fungal spore senses that it is in contact with the plant by the production of small amounts of cutin monomers catalyzed by the low level of cutinase carried by the spore and that these monomers induce the synthesis of cutinase needed for penetration of the fungus into the plant.
Visualization of the Ca
2+
-transport system of the mitotic apparatus of sea urchin eggs with a monoclonal antibody
Authors: Petzelt Christian , Hafner Mathias
Published: May 2006

Monoclonal antibodies have been obtained to components of Ca
2+
-sequestering vesicles from the endoplasmic reticulum of HeLa cells by isolating hybridomas that were generated by the
in vitro
immunization of lymphocytes followed by fusion with plasmocytoma cells. One of these monoclonal antibodies specifically labels punctate structures which appear in the mitotic apparatus of sea urchin eggs at the beginning of prophase and disappear upon the completion of cytokinesis. The antibody inhibits the Ca
2+
uptake of the membrane system
in vitro
. It reacts with one 46-kDa protein out of the complex protein mixture from the membrane fraction. We take all this as evidence that in fact a specific Ca
2+
-transport system is part of the mitotic apparatus, that such a system is very conserved, and that it is most probably derived from the endoplasmic reticulum.
Nucleotide sequence of the human N-myc gene.
Authors: Stanton L W , Schwab M , Bishop J M
Published: May 2006

Human neuroblastomas frequently display amplification and augmented expression of a gene known as N-myc because of its similarity to the protooncogene c-myc. It has therefore been proposed that N-myc is itself a protooncogene, and subsequent tests have shown that N-myc and c-myc have similar biological activities in cell culture. We have now detailed the kinship between N-myc and c-myc by determining the nucleotide sequence of human N-myc and deducing the amino acid sequence of the protein encoded by the gene. The topography of N-myc is strikingly similar to that of c-myc: both genes contain three exons of similar lengths; the coding elements of both genes are located in the second and third exons; and both genes have unusually long 5' untranslated regions in their mRNAs, with features that raise the possibility that expression of the genes may be subject to similar controls of translation. The resemblance between the proteins encoded by N-myc and c-myc sustains previous suspicions that the genes encode related functions.
Conserved chromosomal positions of dual domains of the ets protooncogene in cats, mice, and humans.
Authors: Watson D K , McWilliams-Smith M J , Kozak C , Reeves R , Gearhart J , Nunn M F , Nash W , Fowle J R , Duesberg P , Papas T S
Published: May 2006

The mammalian protooncogene homologue of the avian v-ets sequence from the E26 retrovirus consists of two sequentially distinct domains located on different chromosomes. Using somatic cell hybrid panels, we have mapped the mammalian homologue of the 5' v-ets-domain to chromosome 11 (ETS1) in man, to chromosome 9 (Ets-1) in mouse, and to chromosome D1 (ETS1) in the domestic cat. The mammalian homologue of the 3' v-ets domain was similarly mapped to human chromosome 21 (ETS2), to mouse chromosome 16 (Ets-2), and to feline chromosome C2 (ETS2). Both protooncogenes fell in syntenic groups of homologous linked loci that were conserved among the three species. The occurrence of two distinct functional protooncogenes and their conservation of linkage positions in the three mammalian orders indicate that these two genes have been separate since before the evolutionary divergence of mammals.
Alternative branch points are selected during splicing of a yeast pre-mRNA in mammalian and yeast extracts.
Authors: Ruskin B , Pikielny C W , Rosbash M , Green M R
Published: May 2006

Pre-mRNA splicing in yeast and higher eukaryotes proceeds by similar pathways, in which a probable splicing intermediate and the excised intron are in a lariat configuration. To compare the pre-mRNA splicing mechanisms in yeast and higher eukaryotes, we have analyzed the RNA products resulting from in vitro processing of a yeast intron-containing pre-mRNA in HeLa cell and yeast extracts. In yeast, the RNA branch (2'-5' phosphodiester bond) of the RNA lariat forms at the third adenosine of the TACTAAC box in vivo and in vitro. In contrast, in the HeLa cell extract, the yeast pre-mRNA is accurately spliced, but the RNA lariats contain RNA branches located significantly closer to the 3' splice site than the TACTAAC box. In yeast, mutant pre-mRNAs that lack the TACTAAC box are not spliced in vivo or in vitro. However, these same mutant pre-mRNAs are accurately spliced in the HeLa cell extract. Therefore, although pre-mRNA splicing in yeast and higher eukaryotes proceeds by the same basic pathway, there are substantial differences in the specificity of the biochemical components that mediate the formation of the RNA processing products.
Stochastic response of human blood platelets to stimulation of shape changes and secretion.
Authors: Deranleau D A , Lüthy R , Lüscher E F
Published: May 2006

Stopped-flow turbidimetric data indicate that platelets stimulated with low levels of thrombin undergo a shape transformation from disc to "sphere" to smaller spiny sphere that is indistinguishable from the shape change induced by ADP through different membrane receptor sites and a dissimilar receptor trigger mechanism. Under conditions where neither secretion nor aggregation occur, the extinction coefficients for total scattering by each of the three platelet forms are independent of the stimulus applied, and both reaction mechanisms can be described as stochastic (Poisson) processes in which the rate constant for the formation of the transient species is equal to the rate constant for its disappearance. This observation is independent of the shape assignment, and as the concentration of thrombin is increased and various storage organelles secrete increasing amounts of their contents into the external medium, the stochastic pattern persists. Progressively larger decreases in the extinction coefficients of the intermediate and final platelet forms, over and above those that reflect shape alterations alone, accompany or parallel the reaction induced by the higher thrombin concentrations. The excess turbidity decrease observed when full secretion occurs can be wholly accounted for by a decrease in platelet volume equal in magnitude to the fraction of the total platelet volume occupied by alpha granules. Platelet activation, as reported by the whole body light scattering of either shape changes alone or shape changes plus parallel (but not necessarily also stochastic) alpha granule secretion, thus manifests itself as a random series of transient events conceivably with its origins in the superposition of a set of more elementary stochastic processes that could include microtubule depolymerization, actin polymerization, and possibly diffusion. Although the real nature of the control mechanism remains obscure, certain properties of pooled stochastic processes suggest that a reciprocal connection between microtubule fragmentation and the assembly of actin-containing pseudopodal structures and contractile elements--processes that may exhibit reciprocal requirements for calcium--might provide a hypothetical basis for a rate-limiting step.
Multigene families and vestigial sequences
Authors: Loomis William F. , Gilpin Michael E.
Published: May 2006

Random duplication and deletion events generate complex genomes carrying a large amount of dispensable sequences. We have simulated such events in a computer model. We followed the evolution of a genome carrying at least one copy of each type of gene. Partial duplications and deletions of genes generated nonfunctional vestigial sequences that were dispensable. The size of the genome stabilized only when the amount of dispensable sequences had increased to the point that most deletions did not affect vital genes. Within such genomes, the number of copies of specific genes fluctuated, thereby generating small multigene families. The parameters of the model were tested over 100,000 events in both simple and complex genomes. The results indicate that when the size of the genome is not critical to survival, as appears to be the case within limits in most eukaryotic organisms, the genome carries vestigial sequences that are no longer functional and that many genes are present in multigene families by chance.
Mutations in the E1a gene of adenovirus type 5 alter the tumorigenic properties of transformed cloned rat embryo fibroblast cells.
Authors: Babiss L E , Liaw W S , Zimmer S G , Godman G C , Ginsberg H S , Fisher P B
Published: May 2006

The adenovirus type 5 mutants H5hr1 and H5dl101 contain modifications in the E1a gene affecting the 13S mRNA-encoded 289-amino acid polypeptide and exhibit a cold-sensitive transformation phenotype upon infection of cloned rat embryo fibroblast (CREF) cells. Transformed cell lines expressing solely E1a or E1a and E1b gene products derived from these viruses display enhanced anchorage-independent growth at 37 degrees C versus 32 degrees C and display a cytoskeletal architecture resembling untransformed fibroblastic CREF cells. In contrast, CREF cells transformed by H5wt or the E1a and E1b region of H5wt grow with similar efficiency in agar at 37 degrees C or 32 degrees C and exhibit an epithelioid morphology that is associated with an altered cytoskeleton. Regardless of the expression or presence of other viral early regions, including E1b, E2a, and E4 genes, specific CREF cell lines expressing an altered 289-amino acid protein and a wild-type 12S mRNA-encoded 243-amino acid protein were capable of inducing tumors in nude mice and in immunocompetent syngeneic Fischer rats. In sharp contrast, cells expressing a wild-type 289-amino acid protein were unable to induce tumors in either nude mice or syngeneic rats. The ability to induce tumors did not correlate with alterations in the pattern of viral DNA integration or differential expression of the E1a and E1b genes, nor was the tumor induction a consequence of unique properties of the immortal parental CREF cell line.
Immunological identification of the genes encoding the four myosin heavy chain isoforms of Caenorhabditis elegans.
Authors: Miller D M , Stockdale F E , Karn J
Published: May 2006

The nematode Caenorhabditis elegans produces four distinct myosin heavy chain (MHC) isoforms, A, B, C, and D. The MHC A and MHC B proteins are coordinately expressed in the body wall muscle and are incorporated into different regions of a single kind of thick filament. MHC C and MHC D are exclusively produced in the pharyngeal muscle. Previous studies of mutations that affect MHC B have shown that this isoform is encoded by the unc-54 gene. Three other MHC genes, myo-1, myo-2, and myo-3, were isolated from a C. elegans genomic library by hybridization with fragments of the unc-54 gene. We have now established the MHC isoform encoded by each gene. Restriction fragments from each of these genes were cloned in the plasmid expression vector pUR288, producing fusion proteins between Escherichia coli beta-galactosidase and portions of the MHC rod domains of each gene. The hybrid proteins were screened with a panel of 18 isoform-specific monoclonal antibodies. The results demonstrate that myo-1 encodes MHC D, myo-2 encodes MHC C, and myo-3 encodes MHC A.
Amino acid sequence of human plasma alpha 1B-glycoprotein: homology to the immunoglobulin supergene family.
Authors: Ishioka N , Takahashi N , Putnam F W
Published: May 2006

The complete amino acid sequence has been determined for alpha 1B-glycoprotein (alpha 1B), a protein of unknown function present in human plasma. This protein (Mr approximately equal to 63,000) consists of a single polypeptide chain N-linked to four glucosamine oligosaccharides. The polypeptide has five intrachain disulfide bonds and contains 474 amino acid residues. Analysis of the amino acid sequence by several computer programs shows that alpha 1B exhibits internal duplication and consists of five repeating structural domains, each containing about 95 amino acids and one disulfide bond. alpha 1B has a unique amino acid sequence. However, several domains of alpha 1B, especially the third, show statistically significant homology to variable regions of certain immunoglobulin light and heavy chains. alpha 1B also exhibits sequence similarity to other members of the immunoglobulin supergene family such as the receptor for transepithelial transport of IgA and IgM and the secretory component of human IgA. Because of its internal duplication and its sequence homology to immunoglobulin-like proteins, alpha 1B appears to have evolved from an ancestral gene similar to that of the immunoglobulin supergene family.
RNA-directed RNA polymerases from healthy and from virus-infected cucumber.
Authors: Khan Z A , Hiriyanna K T , Chavez F , Fraenkel-Conrat H
Published: May 2006

Much work has been done on the isolation, purification, and characterization of the RNA-directed RNA polymerase (EC 2.7.7.48) of cucumber mosaic virus (CMV)-infected cucumbers. Uninfected plants were reported to have no such enzyme, but we recently detected low levels of the activity in cucumber. Since tobacco and cowpea contain such an enzyme that is variably increased in amount by various virus (as well as viroid) infections, we assumed that this would also be the case upon CMV infection of cucumber. However, further purification and characterization of the RNA-directed RNA polymerases from healthy and from infected cucumber suggests that these are different enzymes. The presumed CMV replicase was obtained pure and consists of a major polypeptide of Mr 100,000 and minor components of Mr 110,000 and about 10,000. The Km is 5 microM ([3H]GTP) when tobacco mosaic virus RNA is used as template.
Type beta transforming growth factor is the primary differentiation-inducing serum factor for normal human bronchial epithelial cells.
Authors: Masui T , Wakefield L M , Lechner J F , LaVeck M A , Sporn M B , Harris C C
Published: May 2006

Type beta transforming growth factor (TGF-beta) was shown to be the serum factor responsible for inducing normal human bronchial epithelial (NHBE) cells to undergo squamous differentiation. NHBE cells were shown to have high-affinity receptors for TGF-beta. TGF-beta induced the following markers of terminal squamous differentiation in NHBE cells: (i) increase in Ca ionophore-induced formation of crosslinked envelopes; (ii) increase in extracellular activity of plasminogen activator; (iii) irreversible inhibition of DNA synthesis; (iv) decrease in clonal growth rate; and (v) increase in cell surface area. The IgG fraction of anti-TGF-beta antiserum prevented both the inhibition of DNA synthesis and the induction of differentiation by either TGF-beta or whole blood-derived serum. Therefore, TGF-beta is the primary differentiation-inducing factor in serum for NHBE cells. In contrast, TGF-beta did not inhibit DNA synthesis of human lung carcinoma cells even though the cells possess comparable numbers of TGF-beta receptors with similar affinities for the factor. Epinephrine antagonized the TGF-beta-induced inhibition of DNA synthesis and squamous differentiation of NHBE cells. Although epinephrine increased the cyclic AMP levels in NHBE cells, TGF-beta did not alter the intracellular level in NHBE cells in either the presence or absence of epinephrine. Therefore, epinephrine and TGF-beta appear to affect different intracellular pathways that control growth and differentiation processes of NHBE cells.
Directional introgression of mitochondrial DNA in a hybrid population of tree frogs: The influence of mating behavior
Authors: Lamb Trip , Avise John C.
Published: May 2006

A total of 305 individuals from a hybrid population of North American tree frogs was characterized for allozyme and mitochondrial DNA (mtDNA) genotype. Species-specific mating behaviors had suggested the potential for directional hybridization, in which matings between
Hyla cinerea
males and
Hyla gratiosa
females numerically predominate over the reciprocal combination. Such directional bias leads to predictions about expected distributions of the female-transmitted mtDNA markers in F
1
, backcross, and later-generation hybrids. These predictions were fully confirmed by the observed distributions of mtDNA genotypes among these allozymically inferred hybrid classes. Results exemplify the significance of stereotyped mating behaviors in determining the genetic architecture of a hybrid population.
Single-stranded plasmid DNA in Bacillus subtilis and Staphylococcus aureus.
Authors: te Riele H , Michel B , Ehrlich S D
Published: May 2006

Plasmid pC194 was found to exist in a double-stranded and a single-stranded DNA form in Bacillus subtilis and Staphylococcus aureus. This single-stranded DNA was found as a circular molecule of the same size as the parental monomer and corresponded to only one of the two DNA strands. It represented one-third of plasmid copies. Single- and double-stranded DNA copies in similar proportions to the above were detected for five other S. aureus plasmids (pC221, pC223, pE194, pT127, and pT181) and one B. subtilis plasmid (pHV416). S. aureus plasmid pUB110 and Bacillus cereus plasmid pBC16 were, in contrast, predominantly double-stranded.
Disruption of oligosaccharide processing in murine tumor cells inhibits their susceptibility to lysis by activated mouse macrophages.
Authors: Mercurio A M
Published: May 2006

The components of tumor cell surfaces that participate in the recognition and lysis of these cells by activated macrophages have not been identified. One plausible hypothesis is that these components are specific carbohydrate structures. As an initial test of this hypothesis, I have made use of the oligosaccharide processing inhibitors 1-deoxynojirimycin (dNM) and 1-deoxymannojirimycin (dMM). dNM is an inhibitor of the glucosidases involved in the initial steps of oligosaccharide processing. dMM inhibits mannosidase I. P815 cells incubated in the presence of 1-2 mM dNM for 24 hr synthesized mature glycoproteins that contained glucosylated high-mannose asparagine-linked oligosaccharides instead of complex forms. The glucosylated oligosaccharides were present in trypsin digests of the cell surface. The dNM treatment resulted in a diminution in the amount of surface galactose residues as evidenced by neuraminidase/galactose oxidase/NaB3H4 labeling of surface glycopeptides. It did not, however, inhibit protein synthesis or alter the surface polypeptide profile of the tumor cells. P815 and R1- cells incubated in the presence of 1-3 mM dNM for 24 hr were considerably less sensitive to lysis by interferon-gamma-activated macrophages than were cells incubated in control medium. At a dNM concentration of 3 mM, a 71% inhibition of P815 cell lysis was observed. Similarly, P815 and R1- cells incubated in the presence of 2 mM dMM were also less sensitive to macrophage-mediated lysis than were control cells. The inhibitors did not affect cell viability, growth, or gross morphology. These observations suggest that complex asparagine-linked oligosaccharides on tumor cell surfaces may participate in recognition and lysis by activated macrophages.
Therapy with monoclonal antibody to interleukin 2 receptor spares suppressor T cells and prevents or reverses acute allograft rejection in rats.
Authors: Kupiec-Weglinski J W , Diamantstein T , Tilney N L , Strom T B
Published: May 2006

The mouse hybridoma ART 18 monoclonal antibody (mAb), which binds to the rat interleukin 2 (IL-2) receptor, was studied for its effect on heterotopic cardiac allograft survival in two histoincompatible inbred rat strain combinations. Treatment with ART 18 mAb for 10 days after transplantation prolonged allograft survival in a dose-dependent fashion up to about 3 weeks (acute rejection normally occurs within 8 days). ART 18 mAb therapy started at 5 days after transplantation the time of major rejection activity) abrogated acute rejection and extended the survival to about 18 days. The dense cellular infiltrate noted histologically in acute rejection had virtually disappeared after ART 18 mAb treatment. Thus, IL-2 receptor-targeted therapy can be successfully used to prevent and/or treat acute rejection. When spleen cells from antibody-treated recipients bearing well-functioning allografts were adoptively transferred to normal untreated rats that received cardiac allografts 24 hr later, the survival of donor-specific, but not third-party, test cardiac allografts was prolonged significantly; this supports the idea that ART 18 mAb induced "sparing" of suppressor T lymphocytes. Combining infusion of ART 18 mAb with exogenous IL-2-rich conditioned medium produced the same effect as if the mAb alone had been administered, suggesting that an excess of IL-2 does not prevent binding of ART 18 mAb to IL-2 receptor-bearing cells in vivo. These results support the important role of the IL-2 receptor-bearing cells in the mechanism of allograft rejection; they may represent an important target for immunosuppression in clinical organ transplantation.
T-lymphocyte differentiation and the extracellular matrix: identification of a thymocyte subset that attaches specifically to fibronectin.
Authors: Cardarelli P M , Pierschbacher M D
Published: May 2006

A population of murine thymocytes adheres specifically to fibronectin but not to vitronectin, laminin, or collagen type I. The interaction of these thymocytes with fibronectin could be inhibited by the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro, which comprises the previously identified cell-attachment determinant of the molecule, suggesting that the cell attachment site on fibronectin is recognized by these cells. A similar peptide, in which the aspartate residue had been replaced with glutamate, had no effect on this adhesion. The fibronectin-adherent thymocytes were found to be cortisone-sensitive; to bind peanut agglutinin; to have a Thy-1.2+, Ia- surface phenotype; and to express H-2 antigen only weakly on their surface. In addition, approximately 80% of the fibronectin-adherent cells expressed L3T4 and 80% expressed Ly-1 on their surface, whereas greater than 95% were positive for Ly-2. The data suggest that these cells, which constitute 10% of all thymic lymphocytes, are cortical thymocytes. We propose that their adhesion to fibronectin may be important for their differentiation. The binding to fibronectin provides a means to selectively isolate these cells for study.
Human erythrocyte glucose transporter: normal asymmetric orientation and function in liposomes.
Authors: Chen C C , Kurokawa T , Shaw S Y , Tillotson L G , Kalled S , Isselbacher K J
Published: May 2006

The transport function and orientation of the reconstituted human erythrocyte glucose transporter was studied with liposomes made with bovine brain lipid or Escherichia coli lipid. Reconstitution was achieved by a simple octyl glucoside dilution method. The reconstituted transporters with either lipid showed identical counterflow transport activity, the same response to various inhibitors, and characteristic cytochalasin B (CB) labeling. Functional location and purification of the glucose transporter was performed by using gel-permeation high-performance liquid chromatography with octyl glucoside-containing buffer. The reconstituted transport activity was associated only with band 4.5 protein (preactin) and not with band 3 protein. Both CB binding and transport function of the reconstituted transporters were resistant to trypsin but susceptible to chymotrypsin digestion. However, both trypsin and chymotrypsin treatment of unsealed ghosts completely eliminated the CB labeling and transport function of the glucose transporter. In our reconstitution system the glucose transporters maintained a normal asymmetrical (right-side-out) orientation and good transport function. A specific monoclonal antibody against the glucose transporter inhibited CB labeling of the transporters on unsealed ghosts. This was not found with the reconstituted system; however, after freeze-thawing there was a significant inhibition of CB binding by the antibody. These findings suggest that the CB-binding site of the reconstituted transporter is on the inner side of the proteoliposomes.
Human monoclonal antibody directed against an envelope glycoprotein of human T-cell leukemia virus type I.
Authors: Matsushita S , Robert-Guroff M , Trepel J , Cossman J , Mitsuya H , Broder S
Published: May 2006

We report the production and characterization of a human monoclonal antibody reactive against the major envelope glycoprotein of human T-cell leukemia virus type I (HTLV-I), a virus linked to the etiology of adult T-cell leukemia. We exposed lymph-node cells derived from a patient with adult T-cell leukemia to the Epstein-Barr virus in vitro and obtained a B-cell clone (designated 0.5 alpha) by a limiting dilution technique. The secreted product of 0.5 alpha is a monoclonal antibody (also designated 0.5 alpha; that is IgG1 and has kappa light chains) that binds to the cell membrane of T-cells infected with HTLV-I and lyses them in the presence of complement. The antibody does not react with HTLV-I-negative T cells. In electroblot assays, the monoclonal antibody detects a 46-kDa glycoprotein in disrupted HTLV-I virions and a 34-kDa product following digestion of the viral protein with endoglycosidase F. These molecules have been reported to represent the HTLV-I env gene products. The antibody does not react with HTLV-II and HTLV-III virions. Glycoproteins of 61 and 68 kDa, which are known to be encoded at least in part by the env gene of HTLV-I, are precipitated by the antibody from endogenously radiolabeled HTLV-I-infected HUT 102-B2 and MT-2 cells, respectively. These results suggest that this human monoclonal antibody reacts with an env-encoded glycoprotein of HTLV-I. By using a competition assay with a biotin-labeled 0.5 alpha antibody, we observed that 15 out of 15 patients with adult T-cell leukemia had antibodies that block binding of the 0.5 alpha antibody to HTLV-I virions. This suggests that the antigen detected by 0.5 alpha antibody is a common epitope recognized in HTLV-I-infected individuals in vivo. This antibody, as well as the general strategy for making human monoclonal antibodies reactive against pathogenic retroviruses, may have diagnostic or therapeutic application.
Graft-induced behavioral recovery in an animal model of Huntington disease.
Authors: Isacson O , Dunnett S B , Björklund A
Published: May 2006

Bilateral ibotenic acid lesions of the anteromedial neostriatum produce neuropathological and behavioral changes in rats that are characterized by locomotor hyperactivity and severe maze learning impairments, which can be viewed as analogous to changes seen in Huntington disease. Grafts of fetal striatal neurons, implanted either into the lesioned striatum or into the denervated globus pallidus, reduced both the learning impairments and the locomotor hyperactivity, probably via different mechanisms. The results demonstrate the capacity of neural implants for functional neuronal replacement and promotion of functional recovery after damage to a major telencephalic structure participating in complex cognitive and motoric behaviors.
Conditioning-specific membrane changes of rabbit hippocampal neurons measured in vitro.
Authors: Disterhoft J F , Coulter D A , Alkon D L
Published: May 2006

Intracellular recordings were made from hippocampal CA1 pyramidal neurons within brain slices of nictitating membrane conditioned, pseudoconditioned, and naive adult male albino rabbits. All neurons included (26 conditioned, 26 pseudoconditioned, and 28 naive) had stable penetration and at least 60 mV action potential amplitudes. Mean input resistances were approximately equal to 60 mu omega for the three groups. A marked reduction in the afterhyperpolarization (AHP) following an impulse was apparent for conditioned (x = -0.98 mV) as compared to the pseudoconditioned (x = -1.7 mV) and naive (x = -2.0 mV) neurons. The AHP has been attributed previously to activation of a Ca2+-dependent outward K+ current. The distribution of AHP amplitudes for the conditioned group included a new lower range of values for which there was little overlap with the other groups. The conditioning-specific reduction of AHP may be due to reduction of ICa2+-K+ as shown previously for conditioned Hermissenda neurons. This conditioning-induced biophysical alteration of the CA1 pyramidal cell must be stored by mechanisms intrinsic to the hippocampal slice and cannot be explained as a consequence of changes of presynaptic input arising elsewhere in the brain. Our experiments demonstrate the feasibility of analyzing cellular mechanisms of associative learning in mammalian brain with the in vitro brain slice technique.
An unconventional response of adenylate cyclase to morphine and naloxone in the chicken during early development.
Authors: Sakellaridis N , Vernadakis A
Published: May 2006

The developmental profile of basal, NaF- and forskolin-stimulated adenylate cyclase [ATP-pyrophosphatelyase (cyclizing), EC 4.6.1.1] activity was established throughout the 21-day embryonic age of the chicken. The highest activities were observed from day 6 to day 8. Morphine inhibited NaF- and forskolin-stimulated brain adenylate cyclase activities only at days 6-8. The inhibition was not reversed by the antagonist naloxone, which also inhibited the enzyme during the same embryonic period and had no inhibitory effect thereafter. Thus, this action of morphine is not mediated through the conventional opiate receptor-adenylate cyclase system. We propose that the temporal specificity of this effect of morphine may play a role in the development of prenatal opiate effects.
Alterations in calcium content and biochemical processes in cultured skin fibroblasts from aged and Alzheimer donors.
Authors: Peterson C , Goldman J E
Published: May 2006

Aging and Alzheimer disease lead to alterations in several biochemical properties of cultured skin fibroblasts. Total bound calcium increases in fibroblasts due to normal aging (+52%) and is elevated even further with Alzheimer disease (+197%). Processes that require mitochondrial function, such as glucose and glutamine oxidation, declined in cells from aged donors (-25%) and decreased even further in Alzheimer disease (-46%). In addition, biosynthetic processes that depend upon mitochondrial function, such as glucose or glutamine incorporation into protein and lipid, paralleled the oxidative decreases. Cytosolic and nuclear processes such as leucine incorporation into protein and thymidine into DNA were depressed more by aging than Alzheimer disease. These findings suggest that calcium homeostasis and mitochondrial functions are altered more by Alzheimer disease than normal aging.
Antiviral effect of an oligo(nucleoside methylphosphonate) complementary to the splice junction of herpes simplex virus type 1 immediate early pre-mRNAs 4 and 5.
Authors: Smith C C , Aurelian L , Reddy M P , Miller P S , Ts'o P O
Published: May 2006

Selective inhibition of regulatory immediate early (IE) genes of herpes simplex virus type 1 (HSV-1) should inhibit virus growth. Treatment of HSV-1-infected cells with the oligo(nucleoside methylphosphonate) d(TpCCTCCTG) (deoxynucleoside methylphosphonate residues in italic), which is complementary to the acceptor splice junction of HSV-1 IE pre-mRNA 4 and 5, before (1-24 hr) or at the time of infection caused a dose-dependent inhibition in virus replication. Virus titers were decreased 50% and 90% in cells treated with 25 microM and 75 microM oligomer, respectively; at 300 microM, a 99% reduction in virus production was observed. Viral DNA synthesis was reduced 70-75% and there was a 90% reduction in synthesis of viral proteins, including other IE species and viral functional (130-kDa major DNA-binding) and structural (glycoprotein gB) proteins. In the same concentration range, d(TpCCTCCTG) caused a minimal reduction (0-30%) in protein synthesis and growth rates (less than 40%) of uninfected cells. The data suggest that oligo(nucleoside methylphosphonate)s may be effective in antiviral chemotherapy.
Intron insertions and deletions in the beta/gamma-crystallin gene family: the rat beta B1 gene.
Authors: den Dunnen J T , Moormann R J , Lubsen N H , Schoenmakers J G
Published: May 2006

The rat beta B1-crystallin gene is 13.6 kilobases long and contains six exons. The coding region of the gene is divided over five exons. Each functional entity of the protein is encoded by a separate exon except for the carboxyl-terminal extension, which shares the last exon with the fourth protein motif. Exon 2, encoding the amino-terminal extension of the protein, contains two direct repeats with an overall homology of 68% to the rat brain identifier sequence. A copy of the brain identifier sequence is also found in the 3'-flanking region of the gene. The start site of the mRNA was located by S1 nuclease mapping and analysis of the RNA sequence. The 5' end of the gene was shown to be a 27-base-pair noncoding exon, which is separated from the translation start site by 1.36 kilobases of intronic DNA. The 5'-flanking sequence of the beta B1 gene is highly homologous to that of a gamma-crystallin gene.
Identification, nucleotide sequence, and control of developmentally regulated promoters in the hook operon region of Caulobacter crescentus.
Authors: Chen L S , Mullin D , Newton A
Published: May 2006

The major flagellar proteins, including the flagellins and the hook protein, are synthesized periodically in the Caulobacter crescentus cell cycle at the time of flagellum assembly. Although fla genes are regulated at the transcriptional level [Ohta, N., Chen, L.-S., Swanson, E. & Newton, A. (1985) J. Mol. Biol. 186, 107-115], the 5' regulatory regions of C. crescentus genes have not been identified. We describe here the results of nuclease S1 protection assays that map the 5' ends of mRNAs synthesized in vivo from transcription units II (hook operon) and II.1 of the hook gene cluster and locate the corresponding promoter regions PII and PII.1. The two promoters are regulated with different periodicities in the cell cycle and have different genetic requirements for expression. The failure to detect transcripts from either PI or PII in Escherichia coli suggests that developmentally regulated promoters of C. crescentus have different recognition sequences from those of E. coli. There is little nucleotide sequence homology between PII and PII.1. There are, however, three regions of homology between PII and the nucleotide sequence 5' to the 29-kDa-flagellin-related gene, and two of these are in regions of dyad symmetry. We discuss the possibility that DNA-protein interactions at homologous nucleotide sequences like those identified in PII are part of a regulatory gene cascade that participates in timing fla gene expression in the C. crescentus cell cycle.
Role of DNA polymerase alpha and DNA primase in simian virus 40 DNA replication in vitro.
Authors: Murakami Y , Wobbe C R , Weissbach L , Dean F B , Hurwitz J
Published: May 2006

The role of DNA polymerase alpha (pol alpha) and DNA primase has been investigated in the simian virus 40 (SV40) DNA replication system in vitro. Removal of pol alpha and primase activities from crude extracts of HeLa cells or monkey cells by use of an anti-pol alpha immunoaffinity column resulted in the loss of replication activity. The addition of purified pol alpha-primase complex isolated from HeLa cells or monkey cells restored the replication activity of depleted extracts. In contrast, the pol alpha-primase complex isolated from either mouse cells or calf thumus did not. Extracts prepared from mouse cells (a source that does not support replication of SV40) did not replicate SV40 DNA. However, the addition of purified pol alpha-primase complex isolated from HeLa cells activated mouse cell extracts. pol alpha and primase from HeLa cells were extensively purified and separated by a one-step immunoaffinity adsorption and elution procedure. Both activities were required to restore DNA synthesis; the addition of pol alpha or primase alone supported replication poorly. Crude extracts of HeLa cells that were active in SV40 replication catalyzed the synthesis of full-length linear double-stranded (RFIII) DNA in reaction mixtures containing poly(dT)-tailed pBR322 RFIII. Maximal activity was dependent on the addition of oligo(dA), ATP, and creatine phosphate and was totally inhibited by aphidicolin. Since pol alpha alone could not replicate this substrate and since there was no degradation of input DNA, we propose that other enzymatic activities associate with pol alpha, displace the non-template strand, and allow the enzyme to replicate through duplex regions.
A stochastic model of gene-culture coevolution suggested by the "culture historical hypothesis" for the evolution of adult lactose absorption in humans.
Authors: Aoki K
Published: May 2006

A stochastic model of gene-culture coevolution, suggested by the "culture historical hypothesis" of Simoons and McCracken, is presented. According to this hypothesis, adult lactose absorption, believed to be an autosomal dominant trait, attained a high frequency in some human populations due to the positive selection pressure induced by culturally determined milk use in those populations. Two-dimensional Kolmogorov backward equations with appropriate boundary conditions are derived for the ultimate fixation probability of milk users, of the gene for adult lactose absorption, and of both jointly, and for the average time until fixation of the gene. These boundary value problems are solved numerically by the Gauss-Seidel method. I define a theoretical measure of the correlation between gene and culture in terms of the three ultimate fixation probabilities. Monte Carlo simulations are conducted to check and extend the numerical results and also to obtain the first arrival time at gene frequency 0.70, which is approximately the highest observed frequency in any population. Two results that pertain to the culture historical hypothesis are obtained. First, the incomplete correlation observed between adult lactose absorption and milk use does not necessarily constitute evidence against the hypothesis. Second, for the postulated genetic change to have occurred within the 6000-year period since the advent of dairying, either the effective population size was of the order of 100, or, if it was of larger order, the selection coefficient probably had to exceed 5%.
Amyloid fibrils in hereditary cerebral hemorrhage with amyloidosis of Icelandic type is a variant of gamma-trace basic protein (cystatin C).
Authors: Ghiso J , Jensson O , Frangione B
Published: May 2006

A gamma-trace variant protein is the major constituent of the amyloid fibrils in patients from Iceland with hereditary cerebral hemorrhage with amyloidosis. The protein consists of 110 residues and is similar to human urinary gamma-trace basic protein (or cystatin C) beginning at its 11th amino-terminal residue. It has an amino acid substitution (glutamine for leucine) at position 58 (position 68 in gamma-trace numbering), which is near the proposed active site of related proteins--namely, cysteine protease inhibitors and kininogens. It is postulated that a point mutation has occurred, leading to the production of an unusual protein that is abnormally degraded, bound, and/or precipitated. Alternatively, gamma-trace basic protein may be genetically polymorphic, and the variant described here may represent an as-yet-undiscovered isotype or an allelic form that is linked to, but not responsible for, the deposition disease. Our data on the structure of a gamma-trace variant protein suggests that its gene expresses a polyprotein precursor in which active peptides are flanked by basic amino acid residues that permit cleavage to liberate small internal peptides. It is likely that the nucleotide sequence coding for Arg-Xaa and Lys-Xaa repeated several times in the molecule may function as alternative splicing sites for mRNA processing.
Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells.
Authors: Li M L , Aggeler J , Farson D A , Hatier C , Hassell J , Bissell M J
Published: May 2006

When primary mouse mammary epithelial cells are cultured on plastic, they rapidly lose their ability to synthesize and secrete most milk proteins even in the presence of lactogenic hormones, whereas cells cultured on released type I collagen gels show greatly enhanced mRNA levels and secretion rates of beta-casein and of some other milk proteins. We show here that culture on a reconstituted basement membrane from Engelbreth-Holm-Swarm tumor (EHS) allows greater than 90% of cells to produce high levels of beta-casein. By comparison, 30-40% of cells on released type 1 gels and only 2-10% of cells on plastic express beta-casein after 6 days in culture. Because only 40% of cells from late pregnant gland produced beta-casein before culture, the EHS matrix can both induce and maintain an increased level of casein gene expression. Individual basal lamina components were also evaluated. Type IV collagen and fibronectin had little effect on morphology and beta-casein mRNA levels. In contrast, both laminin and heparan sulfate proteoglycan increased beta-casein mRNA levels (1.5- to 4-fold and 2- to 8-fold, respectively). However, for heparan sulfate proteoglycan, increased message was not accompanied by increased secretion of beta-casein. Profound morphological differences were evident between cells cultured on plastic and on EHS matrix, the latter cells forming ducts, ductules, and lumina and resembling secretory alveoli. These results emphasize the vital role of the extracellular matrix in receiving and integrating structural and functional signals that can direct specific gene expression in differentiated tissues.
Ontogenic activation of a fusion gene introduced into sea urchin eggs.
Authors: Flytzanis C N , Britten R J , Davidson E H
Published: May 2006

Regulatory sequences of a sea urchin cytoskeletal actin gene (CyIIIa) were ligated to the bacterial gene coding for chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol O3-acetyltransferase, EC 2.3.1.28) and the construct was injected into unfertilized sea urchin eggs. CAT activity is detected at the early blastula stage, when transcripts of the endogenous CyIIIa gene normally appear. Our measurements show that during activation the amount of CAT enzyme increases at least 100-fold; that there are present in late blastula stage embryos about 5 X 10(5) molecules of CAT mRNA (i.e., approximately 6 times the number of endogenous CyIIIa mRNAs); and that within the range studied the amount of CAT enzyme produced is independent of the number of CyIIIa-CAT genes incorporated per embryo, probably because the genes are present in excess of factors required for their activation. Activation of the CyIIIa-CAT construct is seriously inhibited, or abolished, by successive deletions of upstream CyIIIa sequences.
Induction of DNA synthesis in isolated nuclei by cytoplasmic factors: inhibition by protease inhibitors.
Authors: Wong R L , Gutowski J K , Katz M , Goldfarb R H , Cohen S
Published: May 2006

Cytoplasmic extracts from spontaneously proliferating and mitogen-activated lymphoid cells contain a protein factor called ADR (activator of DNA replication) that induces DNA synthesis in isolated quiescent nuclei. ADR-containing preparations have proteolytic activity, as indicated by their ability to degrade fibrin in a plasminogen-independent and plasminogen-dependent manner. In addition, aprotinin, a nonspecific protease inhibitor, abrogates ADR-induced DNA synthesis in a dose-dependent fashion. Preincubation studies demonstrated that the effect of aprotinin is not due to its suppressive effects on the nuclei themselves. Other protease inhibitors such as leupeptin, p-aminobenzamidine, and N-alpha-tosyllysine chloromethyl ketone are also inhibitory, but soybean trypsin inhibitor is without effect. ADR activity can be removed from active extracts by adsorption with aprotinin-conjugated agarose beads and can be recovered by elution with an acetate buffer (pH 5). These findings are consistent with the interpretation that the initiation of DNA synthesis in resting nuclei may be protease dependent and, further, that the cytoplasmic stimulatory factor we have called ADR may be a protease itself.