bluuebunny/crossref_metadata_2025 huggingface dataset:
A new class of Escherichia coli recBC mutants: implications for the role of RecBC enzyme in homologous recombination.
Authors: Chaudhury A M , Smith G R
Published: May 2006

Mutants of Escherichia coli sensitive to phage T4 gene 2 mutants were obtained following ethyl methanesulfonate mutagenesis. By mapping and complementation analysis, the mutations in each of the six mutants are in recB and recC. By both in vivo and in vitro analyses, the nuclease activity of RecBC enzyme is undetectable in these mutants. However, by several other criteria, such as proficiency in recombination, relative resistance to UV radiation, and viability of the cells in the culture, these mutants are almost identical to their recBC+ parent. The properties of these mutants indicate that the ATP-dependent double-stranded DNA exonuclease activity of RecBC enzyme is not required for recombination. Chi recombinational hotspots, which stimulate recombination by the RecBC pathway, have no detectable activity in the mutants. This result suggests that the nuclease activity of RecBC enzyme is required for Chi activity and is consistent with the hypothesis that Chi stimulates recombination by directing RecBC enzyme to cut DNA at or near Chi.
Both cholera toxin-induced adenylate cyclase activation and cholera toxin biological activity are inhibited by antibodies against related synthetic peptides.
Authors: Jacob C O , Sela M , Pines M , Hurwitz S , Arnon R
Published: May 2006

The immune response against six synthetic peptides corresponding to various segments of the B subunit of cholera toxin was evaluated. Conjugates in which the peptides were covalently linked to tetanus toxoid served for immunization of rabbits. As previously reported, four of these conjugates elicited antibodies cross-reactive with intact cholera toxin. We report here that antisera against two of these synthetic peptides inhibit the entire spectrum of activities of the intact cholera toxin. This is manifested both on the biochemical level (adenylate cyclase induction) and on the biological effect (intestinal fluid secretion). These results indicate that these peptides may serve as suitable candidates for preparation of a synthetic anticholera vaccine.
Expression of the beta-nerve growth factor gene correlates with the density of sympathetic innervation in effector organs.
Authors: Shelton D L , Reichardt L F
Published: May 2006

Although beta-nerve growth factor (NGF), a protein necessary for survival and development of sympathetic neurons, is believed to be a trophic factor that is produced by sympathetic effector organs, its synthesis by these tissues has never been conclusively demonstrated. Using an assay capable of detecting 10 fg of mRNA, we measured the level of NGF mRNA in tissues innervated by sympathetic neurons. NGF mRNA was detected unambiguously in each tissue at a level that appeared to be more than enough to account for the low levels of NGF protein previously detected. Tissues that were densely innervated had comparatively high levels of NGF mRNA, while those with sparser innervation had lower levels. There was a strong positive correlation between the NGF mRNA level and norepinephrine content, a measure of the density of sympathetic innervation. NGF gene expression in one of these tissues, the iris, was shown to be induced by denervation. NGF mRNA was also found in other areas, including elements of the adult peripheral nervous system--the sciatic nerve and the sympathetic and sensory ganglia. In the central nervous system, levels of NGF mRNA were found that are too high to be attributed entirely to the vasculature, suggesting a role for NGF in adult central nervous system function.
Transformation by polyoma virus is drastically reduced by substitution of phenylalanine for tyrosine at residue 315 of middle-sized tumor antigen.
Authors: Carmichael G , Schaffhausen B S , Mandel G , Liang T J , Benjamin T L
Published: May 2006

We used an oligonucleotide to introduce an A----T transversion at nucleotide position 1178 in polyoma virus DNA. The single effect of this mutation is to substitute phenylalanine for tyrosine at residue 315 of the middle-sized tumor (mT) protein (antigen). This site was previously identified as a major phosphate acceptor in the protein kinase reaction of immunocomplexes containing mT antigen. Reconstituted polyoma virus with the transversion, Py-1178-T, produces an altered mT protein that shows about 20% of the activity of wild-type mT antigen in the immunocomplex kinase assay. This residual activity appears to be directed primarily at another tyrosine at position 322 in the mT protein. The transforming ability of Py-1178-T is drastically reduced compared to wild-type virus. The efficiency of transformation by the mutant is less than 1% of that of wild type in focus assays and less than 0.1% in soft-agar growth assays. Cells identified in focus assays with Py-1178-T are generally less transformed in their phenotype than wild-type transformed cells.
Lysosomes are associated with microtubules and not with intermediate filaments in cultured fibroblasts.
Authors: Collot M , Louvard D , Singer S J
Published: May 2006

Double immunofluorescent labeling experiments for lysosomes and either microtubules or vimentin intermediate filaments in cultured well-spread fibroblasts show a remarkable degree of superposition of the lysosomes and the microtubules. Under two different sets of conditions where the microtubules and intermediate filaments are well segregated from one another, the lysosomes remain codistributed with the microtubules. It is suggested that this specific association of lysosomes with microtubules reflects some type(s) of linkage(s) between them and that such linkages may play an important role in the location and intracellular transport of lysosomes inside cells.
Lengths of chromosomal segments conserved since divergence of man and mouse.
Authors: Nadeau J H , Taylor B A
Published: May 2006

Linkage relationships of homologous loci in man and mouse were used to estimate the mean length of autosomal segments conserved during evolution. Comparison of the locations of greater than 83 homologous loci revealed 13 conserved segments. Map distances between the outermost markers of these 13 segments are known for the mouse and range from 1 to 24 centimorgans. Methods were developed for using this sample of conserved segments to estimate the mean length of all conserved autosomal segments in the genome. This mean length was estimated to be 8.1 +/- 1.6 centimorgans. Evidence is presented suggesting that chromosomal rearrangements that determine the lengths of these segments are randomly distributed within the genome. The estimated mean length of conserved segments was used to predict the probability that certain loci, such as peptidase-3 and renin, are linked in man given that homologous loci are chi centimorgans apart in the mouse. The mean length of conserved segments was also used to estimate the number of chromosomal rearrangements that have disrupted linkage since divergence of man and mouse. This estimate was shown to be 178 +/- 39 rearrangements.
Purification to homogeneity and partial characterization of interleukin 2 from a human T-cell leukemia.
Authors: Stern A S , Pan Y C , Urdal D L , Mochizuki D Y , DeChiara S , Blacher R , Wideman J , Gillis S
Published: May 2006

A method utilizing reversed-phase high-performance liquid chromatography has been developed for the purification to homogeneity of interleukin 2 (IL-2) isolated from a human T-cell leukemia. A final purification of 500,000-fold was obtained with a specific activity of pure IL-2 of 10(9) units/mg. The amino acid analysis of natural IL-2 is strikingly similar to the composition deduced from sequence analysis of a cDNA coding for human IL-2. Protein sequence analysis of CNBr-derived peptides yields data consistent with the sequence proposed from cloned cDNA. The availability of homogeneous IL-2 will allow accurate biological studies of its activity free from the contamination of the numerous lymphokine species that are known to be co-produced with IL-2 during the induction procedure.
Divergence in cholesterol biosynthetic rates and 3-hydroxy-3-methylglutaryl-CoA reductase activity as a consequence of granulocyte versus monocyte-macrophage differentiation in HL-60 cells.
Authors: Yachnin S , Toub D B , Mannickarottu V
Published: May 2006

Addition of dimethyl sulfoxide or phorbol myristate acetate (PMA) to HL-60 cell cultures induces granulocytic or monocyte-macrophage differentiation, respectively, in HL-60 cells. Dimethyl sulfoxide-induced granulocyte differentiation in HL-60 cells is associated with a decrease in cellular 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity and with a decrease in the incorporation of [14C]acetate and mevalonate into products of the cholesterol biosynthetic pathway. PMA-induced monocyte-macrophage differentiation in HL-60 cells is associated with a rapid and profound fall in cell proliferation but nonetheless is accompanied by a dose-dependent increase in cellular HMG-CoA reductase activity and [14C]acetate incorporation into the cholesterol biosynthetic pathway. In addition, PMA induces an increase in [14C]mevalonate incorporation into cholesterol and its precursors, suggesting that post-HMG-CoA reductase events in cholesterol biosynthesis are also enhanced. Mature peripheral blood human monocytes possess an active cholesterol biosynthetic pathway, whereas mature human granulocytes are almost entirely lacking in the ability to synthesize post-squalene products. Our results with HL-60 cells indicate that this divergence in sterol-synthesizing ability between two cell lineages, which normally also derive from a common stem cell, can be observed as an early event in the differentiation process.
Phagocytic cells metabolize 25-hydroxyvitamin D3 in vitro.
Authors: Cohen M S , Gray T K
Published: May 2006

Phagocytic cells are widely distributed in tissues known to be important in the metabolism of vitamin D. Incubation of human polymorphonuclear leukocytes and monocytes and resident rat peritoneal macrophages with 3H-labeled 25-hydroxyvitamin D3 leads to the formation of three radioactive peaks. Peak I is most consistent with a lactone derivative of 25-hydroxyvitamin D3, and peak II has been identified as putative 24,25-dihydroxyvitamin D3. Peak III is a novel metabolite of 25-hydroxyvitamin D3 unlike any of the synthetic standards available in our laboratories. Human neutrophils converted more substrate than did the other phagocytes examined. The stimulation of neutrophils by opsonized zymosan or phorbol myristate acetate led to a 4-fold increase in synthesis of the metabolites. These results suggest that vitamin D metabolism by phagocytic cells may play a role in the microenvironmental events that surround bony metabolism and calcium homeostasis.
An immune strain of
Halobacterium halobium
carries the invertible L segment of phage ΦH as a plasmid
Authors: Schnabel Heinke
Published: May 2006

The structure of the circular prophage genome of ΦH varies with high frequency in single colony progeny of the defective lysogen
Halobacterium halobium
R
1
-3. As in linear ΦH DNA, a segment flanked by two copies of the insertion element ISH1.8 is inverted frequently. This L segment can also circularize to a plasmid of 12 kilobase pairs with simultaneous loss of the remaining phage DNA. Strain R
1
-L, which contains this plasmid, is immune to phage infection. A phage variant, ΦHL1, is able to grow on R
1
-L and carries an insertion of 1 kilobase pair in its L segment. ΦHL1 does not grow on normal lysogens. This shows that the plasmid confers to R
1
-L only part of the immunity of normal lysogens.
Nucleotide sequence analysis of the long terminal repeat of human T-cell leukemia virus type II.
Authors: Shimotohno K , Golde D W , Miwa M , Sugimura T , Chen I S
Published: May 2006

The nucleotide sequence of the human T-cell leukemia virus type II (HTLV-II) long terminal repeat (LTR) and its surrounding regions were determined. Our results show the following structural features: (i) the LTR is 763 base pairs (bp) in length and consists of 314 +/- 1 bp of region U3, 248 +/- 1 bp of region R, and 201 bp of region U5; (ii) the terminal nucleotides in the LTR form an inverted repeat of T-G.....C-A; (iii) 6-bp direct repeats of cellular sequences flanking the provirus were present; and (iv) the putative functional signals for initiation or termination of viral RNA synthesis were identified. Comparison of the HTLV-II LTR sequence with that previously published for adult T-cell leukemia virus (ATLV; HTLV-I) shows that the LTRs are distinct. Some small regions are conserved between HTLV-II and ATLV, involving sequences important for transcription and a sequence of 21 nucleotides repeated three times in U3. This 21-bp repeat may be important in regulating viral transcription in lymphoid cells.
A quantitative model for the cooperative mechanism of human hemoglobin.
Authors: Johnson M L , Turner B W , Ackers G K
Published: May 2006

A quantitative model has been developed for the cooperative oxygenation of human hemoglobin. The model correlates the structural and energetic features of ligand-linked subunit interactions within the tetrameric molecule and the coupling of these interactions to the binding of oxygen and Bohr protons. Recent findings are incorporated regarding (i) the sites of regulatory energy change within the tetrameric molecule, (ii) the nature of the Bohr effect for tetramers and dimers, (iii) the fractional Bohr proton release at each stage of oxygenation, (iv) relative probabilities of binding to the alpha and beta chains within the tetramer, and (v) an extensive data base recently obtained on the linked processes of oxygenation, proton binding, and subunit interactions [Chu, A. H., Turner, B. W. & Ackers, G. K. (1984) Biochemistry 23, 604-617]. Least squares minimization was used to evaluate from these data the free energies for the various processes. A special feature of the model lies in the synchronization of Bohr proton release with changes in quaternary structure. This leads to the striking prediction that a major fraction (as much as 30%) of tetramers are in the oxy quaternary structure after the first oxygen is bound. The model provides a rationale for the essential features of regulatory energy control, and it defines several kinds of additional information that are needed for a more complete understanding of the hemoglobin mechanism.
Charge accumulation and photochemistry in leaves studied by thermoluminescence and delayed light emission
Authors: Rutherford A. W. , Govindjee  , Inoue Y.
Published: May 2006

A major breakthrough in our understanding of how plants oxidize water to molecular O
2
was the discovery by P. Joliot and co-workers that the O
2
yield per flash, in a series of light flashes, oscillates with a periodicity of 4. This led to the concept by B. Kok and co-workers that these reactions involve accumulation of four positive charges in independent “O
2
-evolving centers,” which undergo a series of changes in their redox state (the so-called
S
states). In the present paper, we have applied optical techniques (such as thermoluminescence and delayed light emission, both discovered by W. Arnold and co-workers) to monitor charge storage on the O
2
-evolving system in leaves from higher plants. We observed a period of four oscillations in both thermoluminescence and delayed light emission, with maxima on flashes 2 and 6, establishing a relationship with the charge accumulation process in photosynthesis. These measurements provided additional new information: the deactivation of the “O
2
-evolving centers,” which cannot be measured by the O
2
method in the leaves, is in the 20- to 30-s range; and in the dark-adapted leaves, the secondary bound plastoquinone molecule (the so-called secondary electron acceptor
Q
B
) is in equal concentration in its reduced and oxidized forms. The origin of thermoluminescence and delayed light emission, in terms of the recombination of charges on the O
2
-evolving and plastoquinone sides, is also discussed.
Hemizygous interstitial deletion of chromosome 15 (band D) in three translocation-negative murine plasmacytomas.
Authors: Wiener F , Ohno S , Babonits M , Sümegi J , Wirschubsky Z , Klein G , Mushinski J F , Potter M
Published: May 2006

Three murine plasmacytomas that were exceptional in lacking the characteristic (12;15) or (6;15) translocations were studied by G banding and high-resolution banding. One of every two chromosomes 15 (two of four in tetraploid tumors) was shortened in all three tumors. High-resolution banding analysis revealed that this was due to an interstitial deletion in the 15D band region. The two breaks responsible for the deletion have been tentatively localized to the interface of bands D2/3' and within band D2. One of the three plasmacytomas, ABPC45, had a rearranged c-myc gene. All three tumors contained a greater abundance of 2.4-kilobase myc RNA transcripts than normal spleen or thymus. The c-myc gene is located in the 15 D2/3 band region. We suggest that it may have joined the centromeric portion in the deletion plasmacytomas. This transposition may have led to its constitutive activation, as in the more frequent translocation-carrying plasmacytomas.
Regional assignment of the structural gene for human alpha-L-iduronidase.
Authors: Schuchman E H , Astrin K H , Aula P , Desnick R J
Published: May 2006

The structural gene encoding human alpha-L-iduronidase has been assigned to chromosome 22 by using immunologic, electrophoretic, and somatic cell hybridization techniques. Polyclonal rabbit antibodies raised against purified human low-uptake alpha-L-iduronidase were used to discriminate the human and murine isozymes by a sensitive immuno-precipitation assay. The human chromosome constitution of each clone was determined by cytogenetic and enzyme marker electrophoretic techniques. In 65 human (fibroblast)-mouse (RAG) somatic cell hybrids (from four independent fusions), the expression of human alpha-L-iduronidase was 100% concordant with the presence of human chromosome 22; the assignment was confirmed by the demonstration of the human enzyme in tertiary somatic cell hybrids containing only chromosome 22. Further verification of the gene assignment was made by detection of the human enzyme in tertiary chromosome 22 positive hybrids by Ouchterlony immunodiffusion and rocket immunoelectrophoretic experiments with polyclonal anti-human alpha-L-iduronidase antibodies that were monospecific for the human enzyme. Expression of human enzymatic activity in chromosome 22 positive hybrid lines was markedly reduced; for example, a tertiary hybrid (R-G21-J-15), which contained an average of 1.7 chromosome 22s per cell, only had about 15% of the activity detected in normal diploid fibroblasts. Immunologic studies suggested that the reduced expression was due to abnormal post-translational processing or aggregation (or both) of the human and murine isozymes in these hybrids. Regional assignment of the human structural gene to 22pter----q11 was accomplished by gene dosage studies using diploid human fibroblast lines that were partially monosomic or trisomic for chromosome 22.
Effect of ultraviolet radiation on production of epidermal cell thymocyte-activating factor/interleukin 1 in vivo and in vitro.
Authors: Gahring L , Baltz M , Pepys M B , Daynes R
Published: May 2006

UV radiation was found to enhance the release by keratinocytes of epidermal cell thymocyte-activating factor (ETAF), a hormone-like molecule that is physiochemically identical to interleukin 1 (IL-1). This conclusion was supported by the following observations: (i) the keratinocyte cell line PAM 212 retained ETAF/IL-1-producing potential after exposure to UV radiation despite significant loss in cell viability; (ii) epidermal cells from normal and UV radiation-exposed mice were found to produce equivalent amounts of ETAF/IL-1 on a per cell basis with the density of epidermal cells in UV radiation-exposed skin being at least 5-fold above normal values; (iii) under the conditions used, ETAF/IL-1 could be detected in the serum of UV radiation-exposed, but not normal, animals; and (iv) many of the biologic consequences known to be mediated by elevations in ETAF/IL-1--i.e., neutrophilia, elevated levels of complement component 3, serum amyloid P, and plasma fibrinogen--were all observed in animals following a single UV radiation exposure. Animals subjected to chronic UV radiation showed an initial elevation in their levels of acute-phase reactants that returned to normal concentrations within 7 days. This correlates with observations made by others of a "desensitization" to ETAF/IL-1-mediated effects after chronic administration of known exogenous stimulators of inflammatory responses. Further, the UV radiation-induced desensitization took place in spite of demonstrable serum levels of ETAF/IL-1. These results suggest that the mechanism(s) responsible for desensitization is not an inhibition of ETAF/IL-1 synthesis but rather may result from inability of the target cells to perceive this endogenous mediator or to unavailability of serum-associated ETAF/IL-1 for the appropriate targets.
Two different Na,K-ATPases in the optic nerve: cells of origin and axonal transport.
Authors: Specht S C , Sweadner K J
Published: May 2006

Two molecular forms of Na,K-ATPase can be isolated from the central nervous system. The two forms can be distinguished by their sensitivities to cardiac glycosides and by the electrophoretic mobilities of their catalytic subunits, alpha and alpha(+). Because Na,K-ATPase is a membrane-bound enzyme, it would be predicted to move in the rapid phase of axonal transport, and this was used as a means to determine which form(s) is made by a defined neuron of the central nervous system. Retinal ganglion cells were labeled in vivo by intravitreal injection of [35S]methionine; the Na,K-ATPase that was axonally transported down the optic nerve was purified, and the alpha and alpha(+) forms were separated by electrophoresis and detected by fluorography. The two forms were synthesized in the retina in approximately equal amounts. The alpha(+) form was the predominant form transported from the retinal ganglion cells to the lateral geniculate nucleus and superior colliculus. The oligodendrocytes and other sheath cells of the excised optic nerve, in contrast, synthesized only the alpha form when incubated in vitro with [35S]methionine. The labeled Na,K-ATPase found at the nerve endings always included a small amount of the alpha form in addition to the alpha(+) form. The proportions of the two forms did not change with time after transport, and the presence of labeled alpha was not affected by infusion of cycloheximide to inhibit intracranial protein synthesis. Hence, although alpha(+) is the predominant form, the evidence suggests that small amounts of the alpha form are also made and transported by retinal ganglion cells.
Reconstitution of neurotoxin-modulated ion transport by the voltage-regulated sodium channel isolated from the electroplax of Electrophorus electricus.
Authors: Rosenberg R L , Tomiko S A , Agnew W S
Published: May 2006

The functional reconstitution of the voltage-regulated Na channel purified from the electroplax of Electrophorus electricus is described. Reconstitution was achieved by removing detergent with Bio-Beads SM-2 followed by freeze-thaw-sonication in the presence of added liposomes. This preparation displayed heat-stable binding of 3H-labeled tetrodotoxin (TTX) (Kd = 33 nM). 22Na+ influx was stimulated 2- to 5-fold by alkaloid neurotoxins and blocked by TTX. Veratridine activated Na+ influx with a K1/2 of 18 microM, and this activation was blocked by TTX precisely in parallel with specific [3H]TTX binding. Batrachotoxin stimulated 22Na+ flux more effectively than did veratridine. No effect of the peptide anemone toxin II was found. Insertion of the Na channel into membranes resulted in 60-70% of the TTX-binding sites facing the vesicle exterior. Thus, external TTX partially inhibited flux, whereas blockade was complete when TTX was also equilibrated with the vesicle interior. The lipid-soluble local anesthetics tetracaine and dibucaine inhibited flux completely. QX-222, a charged derivative of lidocaine, blocked only a fraction of the channels, apparently those oriented inside-out. Purified samples were predominantly composed of the Mr 260,000-300,000 glycopeptide but contained variable quantities of smaller peptides. Veratridine-dependent flux and peptide compositions were determined on fractions across a gel filtration column profile. Stimulated flux codistributed only with the large peptide.
On the Williams—Watts function of dielectric relaxation
Authors: Shlesinger Michael F. , Montroll Elliott W.
Published: May 2006

It has been observed over the past 15 years that experimental frequency-dependent dielectric constants of broad classes of materials including polymeric systems and glasses may be interpreted in terms of the Williams-Watts polarization decay function [Formula: see text] The exponent α and the time constant
T
depend on the material and fixed external conditions such as temperature and pressure. We derive this form of ϕ
α
(
t
) from the following random-walk model. Suppose that an electric field has been applied for some time to a medium containing many polar molecules (or polar groups in complex molecules) and the direction of their dipole moments remains frozen as the field is removed. Furthermore, suppose that the medium contains mobile defects that on reaching the site of a frozen dipole relax the medium to the degree that the dipole may reorient itself. If the diffusion of defects toward dipoles is executed as a continuous-time random walk composed of an alternation of steps and pauses and the pausing-time distribution function has a long tail of the form ψ(
t
) ∞
t
-1-α
, then the relaxation function has the above fractional exponential form.
Isolation and characterization of the rat tyrosine aminotransferase gene.
Authors: Shinomiya T , Scherer G , Schmid W , Zentgraf H , Schütz G
Published: May 2006

Tyrosine aminotransferase (TAT; L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) from rat liver is subject to glucocorticoid, cAMP, and developmental control. To study the underlying regulatory mechanisms, the TAT structural gene was isolated from a lambda bacteriophage rat DNA library. Heteroduplex analysis revealed that the 2.4-kilobase-long TAT mRNA is encoded by a gene that extends over 11 kilobases and is interrupted by 11 introns. To characterize the presumptive control region, the DNA sequence around the 5' end of the gene was determined and the start site of transcription was identified by nuclease S1 protection experiments. A short sequence homology in an equivalent position relative to the cap site was detected between TAT and tryptophan oxygenase, another glucocorticoid-controlled gene from rat liver. This sequence is related to the sequence 5' T-G-T-T-C-T 3' found in regions of the long terminal repeat of mouse mammary tumor virus, which has been shown to interact with the glucocorticoid receptor [Scheidereit, C., Geisse, J., Westphal, H. M. & Beato, M. (1983) Nature (London) 304, 749-752].
Evidence for the involvement of coupling factor B in the H+ channel of the mitochondrial H+-ATPase.
Authors: Sanadi D R , Pringle M , Kantham L , Hughes J B , Srivastava A
Published: May 2006

Membrane energization by ATP has been measured in vesicles containing purified bovine heart mitochondrial H+-ATPase (ATP synthase) with the voltage-sensitive dye oxonol VI. The dithiol chelator, Cd2+, and the thiol oxidant, copper o-phenanthroline, produced discharge of the membrane potential when added at the steady state and inhibited its establishment when added prior to energization by ATP. These effects, which were reversed by dithiothreitol, were not accompanied by an increase in the nonspecific H+ permeability of the membrane. Passive H+ conduction in proteoliposomes containing F0 (hydrophobic segment of ATP synthase) was assayed by the quenching of 9-aminoacridine fluorescence after establishing a K+ diffusion potential. This conductance was blocked by Cd2+, an inhibitor of coupling factor B (FB). Labeling of F0 with 115Cd2+ at the concentrations that inhibited the F0 conductance followed by gel electrophoresis yielded a single radioactive band with a molecular weight corresponding to FB, the presence of which in the F0 preparation was confirmed by immunoblot staining. The data offer strong evidence that FB is an essential component of the H+ channel of F0, because H+ conduction through the channel is inhibited by chemical modification of FB.
Crambin in phospholipid vesicles: Circular dichroism analysis of crystal structure relevance
Authors: Wallace B. A. , Kohl N. , Teeter M. M.
Published: May 2006

Crambin, a hydrophobic plant seed protein that exhibits sequence homology to membrane-active plant toxins, was incorporated into phospholipid vesicles. Circular dichroism spectroscopy indicates that its structure in vesicles is nearly identical to its structure in 60% ethanol solution, the solvent from which the protein was crystallized. The secondary structure predicted from the circular dichroism data of the ethanol solution closely resembles that determined by x-ray diffraction of the crystals. This agreement suggests that the x-ray structure may form a useful basis for modeling the structure and behavior of lipophilic plant toxins. Finally, because the structure of crambin has been determined in an organic solvent medium, it provides a protein standard for examination of the effect of solvent dipole moment on the circular dichroism spectra of proteins, which may be important for interpretation of data for membrane proteins.
Assembly in vitro of a spanning membrane protein of the endoplasmic reticulum: the E1 glycoprotein of coronavirus mouse hepatitis virus A59.
Authors: Rottier P , Brandenburg D , Armstrong J , van der Zeijst B , Warren G
Published: May 2006

The E1 glycoprotein of coronavirus mouse hepatitis virus A59 was synthesized in vitro by translation of viral mRNA in the presence of dog pancreatic microsomes. Its disposition in the membrane was investigated by digestion with proteases and by selective NH2-terminal labeling. The protein spans the membrane, but only small portions from the NH2 and COOH terminus are exposed respectively in the lumenal and cytoplasmic domains; the bulk of the molecule is apparently buried in the membrane. The protein lacks a cleavable leader sequence and does not acquire its characteristic O-linked oligosaccharides in rough microsomes. It may enter the membrane at any stage during synthesis of the first 150 amino acid residues. These unusual features of the protein might help to explain why it is not transported to the cell surface in vivo but remains in intracellular membranes, causing the virus to bud there.
Artifacts caused by cell microinjection.
Authors: Miller D S , Lau Y T , Horowitz S B
Published: May 2006

The effects of microinjection on Rana pipiens oocytes were determined using cryomicrodissection to measure Na, K, water, and injected radiolabeled sucrose (in gelatin) in the nucleus, animal, and vegetal ooplasm and injected bolus (reference phase, RP). The results point to potential problems in the interpretation of microinjection experiments. When oocytes were injected and incubated in Ringer's solution, nucleus, ooplasm, and RP lost K and sucrose and gained Na. Patterns of loss and gain were complex but were consistent with continuous solute leakage at the injection site causing artifactual intracellular diffusion gradients. In spite of leakage, oocytes completed scheduled meiotic maturation when exposed to progesterone. When oocytes were microinjected and incubated in paraffin oil (a medium in which polar solutes cannot exchange), nuclear and ooplasmic Na, K, and water concentrations remained identical to those in uninjected cells. Neither microinjection per se nor the injected bolus affected intraoocytic solute distributions. These findings imply that, after microinjection in aqueous media, metabolites are lost from and redistribute in cells, and that these artifactual changes are inadequately reflected in the ability of the cell to carry out a complex process. They also show that injection artifacts can be avoided by injecting and incubating cells under paraffin oil.
Insertion of retrovirus into the first intron of alpha 1(I) collagen gene to embryonic lethal mutation in mice.
Authors: Harbers K , Kuehn M , Delius H , Jaenisch R
Published: May 2006

Integration of the Moloney murine leukemia virus (M-MuLV) into the germ line of Mov-13 mice blocked formation of stable alpha 1(I) collagen mRNA and led to an embryonic lethal mutation. A 14-kilobase fragment representing the integration site of the virus was molecularly cloned and identified as the alpha 1(I) collagen gene. Sequence and nuclease S1 mapping analyses were performed to characterize the position of the proviral genome in relation to the transcriptional map of the mutated gene. The results indicated that the virus has inserted into the first intron 19 base pairs downstream of the intron/exon boundary. Sequence comparison showed a striking homology of exon sequences and sequences up to 215 base pairs upstream of the mRNA start between the mouse and the human alpha 1(I) collagen gene. This indicates that the sequences upstream of the mRNA start are highly conserved during evolution, suggesting that this region has an important role in the control of tissue-specific collagen expression.
Isolation and partial characterization of concanavalin A receptors on cloned cytotoxic T lymphocytes.
Authors: Sitkovsky M V , Pasternack M S , Lugo J P , Klein J R , Eisen H N
Published: May 2006

A small set of concanavalin A (Con A)-binding glycoproteins was isolated from the surface membrane of cloned cytotoxic T lymphocytes (CTL) and partly identified using monoclonal antibodies. The binding of Con A by these glycoproteins on the CTL surface results in the secretion of gamma-interferon and in blocking the effector functions of the cells-namely, antigen-specific and lectin-dependent cytotoxicity. The Con A is evidently bound tightly to some surface structures ("Con A-receptors") that are required for the activation and cytotoxic activity of CTL. To isolate and identify these receptors, antibodies to Con A were used. After Con A was allowed to bind to radiolabeled cloned CTL (labeled with 125I or [35S]methionine or 3H-labeled amino acids), the cells were washed thoroughly, lysed in detergents and anti-Con A antibodies were added to bind to the Con A-receptor complexes. The resulting aggregates were adsorbed with protein A-bearing Staphylococci and the receptors were then specifically released from the pelleted bacteria by alpha-methyl-D-mannoside and analyzed by polyacrylamide gel electrophoresis under reducing conditions. Eight to nine labeled components were seen by autoradiography and with the aid of monoclonal antibodies to known T-cell surface molecules, four were identified as T200, lymphocyte function-associated antigen (LFA)-1, alpha- and beta-chains, and (on some clones) Lyt-2. Other components with Mr congruent to 160,000, 120,000, 46,000, 42,000, and 23,000 have not been identified. The procedures described here may have general application in the studies of the functional properties of other cell surface molecules.
Axonal transport of a subclass of tau proteins: evidence for the regional differentiation of microtubules in neurons.
Authors: Tytell M , Brady S T , Lasek R J
Published: May 2006

Tubulin, the major constituent of microtubules, is anterogradely transported within the axon as part of slow component a (SCa; 0.2-1.0 mm/day). This raises the possibility that the microtubule-associated proteins (MAPs) may be transported at the same rate. To examine this question, the high molecular weight and tau MAPs obtained from whole brain preparations of microtubules were compared with the proteins of SCa in guinea pig retinal ganglion cell axons by using phosphocellulose chromatography and one- and two-dimensional polyacrylamide gel electrophoresis. Only two of the tau proteins were found to be cotransported with axonal tubulin, although four tau and two high molecular weight MAPs were synthesized in the retina. This result suggests either that the retinal ganglion cell synthesizes only those two tau proteins or that it synthesizes several of the MAPs, but commits to axonal transport just two of the tau proteins. In either case, these observations are consistent with the transport of intact microtubules and demonstrate that axonal microtubules represent a distinct subset of brain microtubules. Such a distinction may be related to unique properties of the axonal cytoskeleton.
Possible ideal lac operator: Escherichia coli lac operator-like sequences from eukaryotic genomes lack the central G X C pair.
Authors: Simons A , Tils D , von Wilcken-Bergmann B , Müller-Hill B
Published: May 2006

Five DNA fragments have been cloned from yeast, chicken, and mouse DNA that titrate lac repressor in an Escherichia coli lac+ I+Z+ wild-type strain when on a multi-copy plasmid. The five repressor-binding sequences have been identified by DNA sequence determinations and DNase cleavage-inhibition patterns. They share the 14-base-pair symmetrical consensus sequence 5' T-G-T-G-A-G-C:G-C-T-C-A-C-A 3' (the colon represents the center of symmetry), which is an inverted repeat of 7 base pairs of the left half of the E. coli lac operator. A similar perfect palindromic DNA fragment--an 11-base-pair inverted repeat of the left half of the lac operator--was synthesized. The cloned synthetic DNA 5' G-A-A-T-T-G-T-G-A-G-C:G-C-T-C-A-C-A-A-T-T-C 3' binds lac repressor 8-fold more tightly than does wild-type E. coli lac operator DNA.
T-cell growth factor: complete nucleotide sequence and organization of the gene in normal and malignant cells.
Authors: Holbrook N J , Smith K A , Fornace A J , Comeau C M , Wiskocil R L , Crabtree G R
Published: May 2006

Using a cloned cDNA copy of T-cell growth factor (TCGF) mRNA from the Jurkat leukemic T-cell line, we have isolated three overlapping TCGF genomic clones from a human DNA library. The entire TCGF gene is contained within two adjacent EcoRI fragments spanning about 8 kilobases. The complete nucleic acid sequence was determined. The gene is divided into four exons. The 5' untranslated region and the first 49 amino acids of the protein, 20 of which constitute a signal polypeptide and are not present in the secreted protein, are encoded by the first exon. Exons 2 and 3, separated from each other by a long intervening sequence, contain coding information for the next 20 and 48 amino acids, respectively. The remaining 36 amino acids and the 3' untranslated region are contained in the fourth exon. A promoter sequence T-A-T-A-A-A is present 77 base pairs (bp) upstream from the translation initiation site, and a CAT homology region occurs 104 bp upstream from the initiation site. A putative site for initiation of mRNA transcription was identified 53 bp 5' of the translation initiation codon. The organization of the gene was shown by Southern blot analysis to be identical in normal peripheral blood lymphocytes and in a variety of malignant lymphoid cell types. Restriction analysis of these cellular DNAs produced results exactly as predicted by the map for the cloned genomic TCGF, indicating that there is only a single copy of the human TCGF gene.
Progesterone receptor subunits are high-affinity substrates for phosphorylation by epidermal growth factor receptor.
Authors: Ghosh-Dastidar P , Coty W A , Griest R E , Woo D D , Fox C F
Published: May 2006

Purified preparations of epidermal growth factor (EGF) receptor were used to test hen oviduct progesterone receptor subunits as substrates for phosphorylation catalyzed by EGF receptor. Both the 80-kilodalton (kDa) (A) and the 105-kDa (B) progesterone receptor subunits were phosphorylated in a reaction that required EGF and EGF receptor. No phosphorylation of progesterone receptor subunits was observed in the absence of EGF receptor, even when Ca2+ was substituted for Mg2+ and Mn2+. Phospho amino acid analysis revealed phosphorylation at tyrosine residues, with no phosphorylation detectable at serine or threonine residues. Two-dimensional maps of phosphopeptides generated from phosphorylated 80- or 105-kDa subunits by tryptic digestion revealed similar patterns, with resolution of two major, several minor, and a number of very minor phosphopeptides. The Km of progesterone receptor for phosphorylation by EGF-activated EGF receptor was 100 nM and the Vmax was 2.5 nmol/min per mg of EGF receptor protein at 0 degrees C. The stoichiometry of phosphorylation/hormone binding for progesterone receptor subunits was 0.31 at ice-bath temperature and approximately 1.0 at 22 degrees C.
Endonuclease S1-sensitive site in chicken pro-alpha 2(I) collagen 5' flanking gene region.
Authors: Finer M H , Fodor E J , Boedtker H , Doty P
Published: May 2006

A site that is preferentially cleaved by the single-strand-specific endonuclease from Aspergillus oryzae was located in vitro 180 base pairs upstream from the 5' end of the chicken pro-alpha 2(I) collagen gene. It is found in supercoiled plasmids with a negative superhelical density of -0.024 or more but not in linear DNA molecules. The nuclease S1 sensitivity is retained in plasmids containing genomic fragments extending from position +8 to -285 (where +1 is the first transcribed base) and from -147 to -351 and also in a 5.7-kilobase EcoRI fragment that extends 1.6 kilobases 5' and 4.1 kilobases 3' to the 5' end of the gene. Analysis at the nucleotide level on a DNA sequence gel places the site at -181 to -182 on the sense strand and at -182 to -184 and -192 to -195 on the nonsense strand. These sites lie within a stretch of 42 pyrimidines interrupted by a single guanine and within the sequence T-C-C-C-T-C-C-C-T-T-C-C-T-C-C-C-T-C-C-C-T.
Nucleotide sequence surrounding transcription initiation site of xylABC operon on TOL plasmid of Pseudomonas putida.
Authors: Inouye S , Ebina Y , Nakazawa A , Nakazawa T
Published: May 2006

The xylABC operon on the TOL plasmid directs the synthesis of enzymes for conversion of toluene to benzoate and is positively controlled by the regulatory gene xylR. In the study here the nucleotide sequence was determined for the regulatory region of this operon. The in vivo transcription initiation site of the operon was determined by S1 nuclease and reverse transcriptase mapping. RNA was prepared from m-methylbenzyl alcohol-induced cells of Pseudomonas putida and Escherichia coli carrying pTN2, a derivative of the TOL plasmid containing the structural and regulatory genes of the entire toluene-degrading pathway. The amount of E. coli mRNA was estimated to be only 10% of that of P. putida mRNA. Consensus sequences of the -10 region (Pribnow box) and the -35 region (RNA polymerase recognition site) in E. coli genes were not found in the region preceding the transcription initiation site, whereas a sequence complementary to the 3' end of the 16S rRNA of Pseudomonas aeruginosa and E. coli existed in front of the predicted start codon of the xylA gene. These results explain the inefficient expression of TOL genes in E. coli.
Shared idiotopes among monoclonal antibodies specific for A/PR/8/34 (H1N1) and X-31(H3N2) influenza viruses.
Authors: Moran T , Liu Y C , Schulman J L , Bona C A
Published: May 2006

A monoclonal antibody specific for the hemagglutinin (HA) of influenza A X-31 (H3N2) virus (X-31) was obtained during a fusion of spleen cells from a BALB/c mouse immunized with influenza A/PR/8/34 (H1N1) virus (PR8). This monoclonal antibody (Py206) shares crossreactive idiotopes expressed on several monoclonal antibodies specific for PR8 HA and X-31 HA as well as an individual idiotope shared with one monoclonal antibody specific for X-31 HA. The presence of shared idiotypy among antibodies of such diverse binding specificities may result from regulation by idiotype-specific T cells. In addition, the results suggest that, in contrast to the diversity of paratypes expressed, relatively few germ-line variable region genes may be used in the antibody response to influenza A virus antigens.
Schwann cell expression of a major myelin glycoprotein in the absence of myelin assembly.
Authors: Poduslo J F , Berg C T , Dyck P J
Published: May 2006

Quiescent Schwann cells in the distal segment of the permanently transected nerve produced basal levels of the major myelin glycoprotein, P0, in the absence of myelin assembly. Low levels of P0 could be detected at 35 days after transection by autoradiographic analysis of radioiodinated lectin binding after protein separation by high-resolution sodium dodecyl sulfate pore gradient electrophoresis and by fluorographic analysis after electrophoresis of [3H]fucose- and [3H]mannose-labeled glycoproteins after incorporation into endoneurial slices. Immunoreactivity to P0 in the transected nerve could also be demonstrated with antisera against P0 as evaluated by direct "immune overlay" after electrophoresis. These results indicate that the requirement for continuing signals from appropriate axons to make detectable amounts of myelin-specific proteins and glycolipids is not absolute. Schwann cells, therefore, like oligodendrocytes, can synthesize myelin components in the absence of neuronal influence, although information from neuronal elements probably is required for myelin assembly by Schwann cells and for myelin compaction by oligodendrocytes.
Alteration of lipid-phase behavior in multiple sclerosis myelin revealed by wide-angle x-ray diffraction.
Authors: Chia L S , Thompson J E , Moscarello M A
Published: May 2006

Wide-angle x-ray diffraction studies revealed that the lipid-phase transition temperature of multiple sclerosis (MS) myelin was about 20 degrees C lower than that of normal myelin, indicating differences in the physical organization of the bilayer. The transition temperature of liposomes prepared from total lipid extracts of normal myelin was 12 degrees C lower than that for corresponding intact myelin, demonstrating that the protein of normal myelin had a substantial ordering effect on the lipid bilayer. The transition temperature for liposomes of MS myelin lipid was essentially similar to that for isolated MS myelin. Because the protein/phospholipid ratio was higher in MS myelin, and no difference in degree of fatty acid saturation was observed, the inability of MS myelin protein to organize the lipid reflects a qualitative difference in the proteins.
Evidence for a human histone gene cluster containing H2B and H2A pseudogenes.
Authors: Marashi F , Prokopp K , Stein J , Stein G
Published: May 2006

Not all members of the human histone gene family are functional. We have isolated a human H2B pseudogene that contains alterations in the protein-coding sequences as well as in the 3' and 5' flanking sequences that preclude expression of a functional H2B histone protein. There are three modifications in the amino acid-coding region: a single-base deletion producing a frame shift, a single-base substitution resulting in a codon change from serine to tryptophan (an amino acid not present in histones), and the absence of a stop codon. Analysis of nucleotide sequences upstream from the AUG start signal indicates the absence of a "TATA" box and other putative consensus regulatory sequences. In the 3' flanking region, a highly conserved block of 22 nucleotides that exhibits hyphenated dyad symmetry is displaced downstream. Within the same genomic segment, the adjacent H2A histone gene is missing 12 nucleotides, resulting in a deletion of four amino acids in a highly conserved region of the protein.