bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti-idiotypic route to anti-acetylcholine receptor antibodies and experimental myasthenia gravis.
Authors: Wassermann N H , Penn A S , Freimuth P I , Treptow N , Wentzel S , Cleveland W L , Erlanger B F
Published: May 2006

trans-3,3'-Bis[alpha-(trimethylammonio)methyl]azobenzene bromide (BisQ) is a potent agonist of the acetylcholine receptor (AcChoR) of Electrophorus electricus. BisQ is highly constrained, suggesting that its structure is complementary to the combining site of the AcChoR when the latter is in its activated state. Antibodies produced in rabbits to a conjugate of bovine serum albumin and a derivative of BisQ mimicked the binding characteristics of the AcChoR with respect to the order of binding of a variety of agonists and to the preferred recognition of decamethonium ion (an agonist) over hexamethonium ion (an antagonist). Immunization of three rabbits with purified anti-BisQ yielded antisera having binding characteristics of anti-AcChoR in that, by complement fixation and enzyme immunoassay, crossreactions with receptor preparations from rat, Torpedo, and eel could be demonstrated in sera of all three rabbits immunized. Two of the three rabbits showed signs of muscle weakness similar to that seen after immunization with the AcChoR. One of the rabbits was injected intramuscularly with neostigmine and showed temporary improvement. Another showed post-tetanic exhaustion of hind-limb muscles after stimulation of the sciatic nerve at 50 Hz. Antibodies reactive with the AcChoR, therefore, were elicited by immunization with an antibody to a potent ligand of the AcChoR without the necessity of isolating the receptor itself. A similar mechanism may play a part in the etiology of at least some autoimmune diseases in which antibodies to various other receptors are involved.
Prostaglandins E1 and E2 interact with prostaglandin F2alpha to regulate initiation of DNA replication and cell division in swiss 3T3 cells.
Authors: Otto A M , Nilsen-Hamilton M , Boss B D , Ulrich M O , Jimenez De Asua L
Published: May 2006

Prostaglandin (PG) E1 or E2 added at 2-1,000 ng/ml to quiescent cultures of Swiss 3T3 cells synergistically enhanced the rate of initiation of DNA replication stimulated by PGF2 alpha alone or with insulin. Neither PGD2 nor PGF1 alpha had any effect with PGF2 alpha. An increase in the rate of entry into S phase also occurred when PGE1 or PGE2 was added 8 or 15 hr after addition of PGF2 alpha. However, adding PGE1 and PGE2 together with PGF2 alpha did not further enhance the synergistic effect observed with PGE1 or PGE2 separately. The synergistic effect was also observed in stimulation of 2-deoxyglucose uptake but not in early changes of intracellular levels of cAMP. These results may be relevant in understanding the control of fibroblastic proliferation in wound healing and may provide an alternative mechanism for oncogenic transformation.
Identity of human epidermal growth factor (EGF) receptor with glycoprotein SA-7: evidence for differential phosphorylation of the two components of the EGF receptor from A431 cells.
Authors: Carlin C R , Knowles B B
Published: May 2006

A 165-kilodalton (kDal) surface glycoprotein encoded by human chromosome 7 (SA-7) had been characterized by using antisera raised against human chromosome 7-containing somatic cell hybrids. We now present evidence that SA-7 is the human receptor for epidermal growth factor (EGF) and that these antisera recognize human-specific determinants. The gene coding for the human EGF receptor is localized to the p12 to p22 region of chromosome 7. We have characterized the 145-kDal/165-kDal EGF receptor doublet of A431 cells after immunoprecipitation of radiolabeled cell extracts with these antisera. We find that a protein with endogenous kinase activity copurifies with the A431 receptor doublet and that both components of the doublet contain phosphotyrosine and phosphothreonine and the 165-kDal component contains phosphoserine as well. Further, although each component of the receptor doublet has an average pI of 7, both display charge heterogeneity and appear to have unique charge isomers. The relationship between the two components of the A431 EGF receptor is discussed.
Methylation of simian virus 40 Hpa II site affects late, but not early, viral gene expression.
Authors: Fradin A , Manley J L , Prives C L
Published: May 2006

DNA methylation has been correlated with reduced gene expression in a number of studies, although evidence for a casual link between the two events has been lacking. Because microinjection of simian virus 40 (SV40) DNA into the nucleus of Xenopus laevis oocytes results in the synthesis of both early and late viral gene products, it was possible to test whether a specific methylation event can affect gene expression. The single SV40 Hpa II site at 0.72 SV40 map units was specifically methylated with Hpa II methylase. When this DNA was injected into oocytes, there was a marked reduction in the synthesis of the major late viral capsid protein VP-1, relative to the synthesis by an unmethylated control. However, production of the early proteins (the large and small tumor antigens) was not affected by Hpa II methylation. Therefore, methylation at a single site on the viral DNA located near the 5' end of the late region can specifically repress late gene expression. The possible mechanisms by which this repression is mediated are discussed.
Electron microscopic study of natural killer cell-tumor cell conjugates
Authors: Frey Tom , Petty Howard R. , McConnell Harden M.
Published: May 2006

Natural killer (NK) cells were obtained from C3H mouse spleens according to a modified version of the method of Kuribayashi
et al.
[Kuribayashi, K., Gillis, S., Dern, D. E. & Henney, C. S. (1981)
J. Immunol
. 126, 2321-2327]. These cells retain
in vitro
cytotoxicity against certain model tumor cell targets and appear homogeneous by morphological criteria. NK cells, YAC (tumor) cells, and NK cell-YAC cell conjugates have been examined with scanning (SEM) and transmission (TEM) electron microscopy. SEM experiments have shown that: (
i
) NK cells are large and possess various shapes in contrast to the YAC target cells which are smaller and round, (
ii
) YAC cells have uniformly distributed microvilli whereas the NK cell microvilli are most prominent in the area of effector-to-target contact, and (
iii
) in the absence of target cells, NK cell microvilli are found in a small number (usually 1-3) of cell surface locations. The region of NK cell-tumor cell contact has also been examined with TEM. The cells were stained with ruthenium red/OsO
4
. The electron-dense ruthenium red/OsO
4
reaction product was consistently found in regions of close cell-cell contact, suggesting that carbohydrates were not completely cleared from areas of contact and that target and effector membranes do not fuse extensively. TEM observations indicate that NK cells have structurally unique granules. The granules are composed of at least two distinct compartments. The outer compartment contains the lysosome-associated enzymes acid phosphatase and inorganic trimetaphosphatase. No enzymatic activities have been found associated with the inner compartment. NK cells appear to degranulate when incubated with YAC cells. Under those circumstances, limited areas of the NK cytoplasm contain vacuole-like areas possessing granules and apparent granular debris. Degranulation appears to be involved in the cytotoxic function of NK cells.
Protection against syngeneic lymphoma by a long-lived cytotoxic T-cell clone.
Authors: Dailey M O , Pillemer E , Weissman I L
Published: May 2006

The effect of a cloned T-cell line on the in vivo growth of syngeneic lymphoma cells was studied. 1E4 is an H-2-restricted cytotoxic T-cell clone that efficiently kills Abelson virus-induced lymphoma target cells (L1-2) at low effector/target ratios, as measured by in vitro cytotoxicity assays. In addition, it is long lived in vitro in the absence of stimulation and survives for more than 1 wk in vivo in the absence of exogenous antigen or growth factors. Mice injected intraperitoneally with lethal doses of L1-2 and then treated with 1E4 survived longer than animals treated with saline or with a control T-cell clone. Multiple weekly injections of effector cells, or a single injection in animals given a low dose of tumor cells, resulted in 50-80% long-term survivors. The observation that intravenous injection of killer cells was less effective than intraperitoneal treatment, coupled with the previous demonstration of markedly abnormal circulatory patterns for T-cell clones, suggests that those animals succumbing to progressively growing neoplasm die because the effector cells are unable to home into peripheral sites of tumor deposition. Thus, although this cytotoxic T-cell clone does have useful in vivo activity, its function may be partially limited by a generalized defect in migration.
Specific induction of erythroleukemia and myelogenous leukemia in Sprague-Dawley rats.
Authors: Huggins C B , Grand L , Ueda N
Published: May 2006

The experimental induction of leukemias of two sorts by two powerful chemical carcinogens was investigated in rats of a single strain. In Sprague-Dawley rats a series of intravenous injections of N-nitroso-N-methylurea selectively elicited myelogenous leukemia in high yields, whereas erythroleukemia was not evoked. Conversely, a set of intravenous injections of 7,8,12-trimethylbenz[a]anthracene specifically elicited erythro-leukemia in high incidence in the rats, whereas myelogenous leukemia was not produced.
Characterization of a monoclonal antibody to human erythropoietin.
Authors: Weiss T L , Kavinsky C J , Goldwasser E
Published: May 2006

Hybrid cells that synthesize a monospecific antibody directed toward erythropoietin have been produced by the fusion of mouse plasmacytoma cells with spleen cells from rats immunized against human erythropoietin. The antibody binds the alpha and beta forms and the asialo alpha form of erythropoietin to the same extent. It is an immunoglobulin of the IgG class and binds only erythropoietin in an impure preparation of the hormone. Biologically active unlabeled erythropoietin competes with biologically inactive radiolabeled hormone for monoclonal antibody binding sites. In addition, the biological activity of erythropoietin measured in vitro is not inactivated when it is bound by the monoclonal antibody.
Optic cup formation: a calcium-regulated process.
Authors: Brady R C , Hilfer S R
Published: May 2006

Invagination of the optic vesicle to form the optic cup is an important event in the formation of the eye in the early embryo. To obtain support for earlier conclusions that a contractile process is involved, calcium dependency of optic cup formation was tested. Heads were excised from chicken embryos at the optic vesicle stage of development (stage 13) and incubated in nutrient medium containing antagonists or agonists of calcium transport. Invagination was reversibly inhibited by the Ca2+ antagonists verapamil and papaverine. It was initiated in a precocious fashion by the Ca2+ ionophore A23187 but only in the presence of external Ca2+. Neither caffeine, theophylline, nor A23187 (in the absence of external Ca2+) were able to initiate precocious optic cup formation. Trifluoperazine and chlorpromazine reversibly inhibited optic cup formation while chlorpromazine sulfoxide had no effect at the concentrations used. The binding of [3H]trifluoperazine to isolated stage 13 heads revealed a class of Ca2+-dependent binding sites having a Kd similar to that of calmodulin. These results indicate a Ca2+-dependence for optic cup formation and that the source of the Ca2+ may be extracellular. This Ca2+ dependence probably is conferred to the system by calmodulin.
Human antibody to OFA-I, a tumor antigen, produced in vitro by Epstein-Barr virus-transformed human B-lymphoid cell lines.
Authors: Irie R F , Sze L L , Saxton R E
Published: May 2006

We established two long-term human B-lymphoblastoid cell lines (L55 and L72) transformed by Epstein-Barr virus that produced IgM kappa antibodies to the human tumor antigen, OFA-I. Periphral blood lymphocytes obtained from melanoma patients were used as the source of the B lymphocytes. Antibody specificity was determined by the immune adherence assay using various human cancer and noncancer tissues as targets. L55 antibody (designated anti-OFA-I-1) reacted with a variety of human tumor types whereas L72 antibody (designated anti-OFA-I-2) reacted only with tumor cells of neuroectodermal origin (melanoma, glioma, and neuroblastoma). The levels of IgM detected in the spent medium of 1 X 10(6) L55 and L72 cells were 4 and 9 micrograms/ml, respectively, by radioimmunoassay.
A processed human immunoglobulin epsilon gene has moved to chromosome 9.
Authors: Battey J , Max E E , McBride W O , Swan D , Leder P
Published: May 2006

Processed genes--genes that resemble processed RNA transcripts rather than interrupted genomic sequences--have been identified as dispersed members of several gene families. Here we describe a processed gene that is one of the three human IgE-like sequences present in the human genome. The processed IgE gene has precisely lost its three intervening sequences, thereby fusing its four coding domains. The homology of the gene to its functional counterpart ends in an adenine-rich tail followed by an 11-base-pair sequence that is directly repeated 150 base pairs 5' to its first coding domain. In addition, the processed gene is located on human chromosome 9 rather than on chromosome 14, the site of the active immunoglobulin locus. The structure and evident mobility of this sequence support the concept that sequences can move about in the genome via RNA intermediates and that processed genes are a prominent feature of genomic structure.
Orientation of cohesive end site cos determines the active orientation of chi sequence in stimulating recA . recBC-mediated recombination in phage lambda lytic infections.
Authors: Kobayashi I , Murialdo H , Crasemann J M , Stahl M M , Stahl F W
Published: May 2006

Sequence chi, 5'G-C-T-G-G-T-G-G, locally enhances homologous recombination by recA and recBC proteins of Escherichia coli. Previous work showed that, in phage lambda, chi is more active in one orientation (leftward) than in the other (rightward). Inverting cos, the sequence for the mature DNA ends of lambda, reverses this orientation dependence: the rightward chi becomes fully active, and the leftward chi becomes relatively inactive. We surmise that chi action in phage lambda is coupled with DNA packaging or injection.
Ultrastructure of a hyaluronic acid matrix
Authors: Hadler Nortin M. , Dourmashkin Robert R. , Nermut Milan V. , Williams Lynn D.
Published: May 2006

Freeze-etch replicas of a hylauronic acid matrix were visualized by electron microscopy. In water a coarse branching fibrillar network of hyaluronic acid aggregates was seen. The high solvent permeability of this matrix suggests that the spaces observed are relatively devoid of unaggregated polymer. Addition of calcium disordered the matrix, resulting in a more dispersed felt of polymer.
Covalent adducts of DNA and the nonprotein chromophore of neocarzinostatin contain a modified deoxyribose.
Authors: Povirk L F , Goldberg I H
Published: May 2006

When the nonprotein chromophore of neocarzinostatin was allowed to react with either calf thymus DNA or poly(dA-dT) . poly(dA-dT) in the presence of 2-mercaptoethanol and the DNA was precipitated with ethanol, 5% of the fluorescence attributable to the naphthalene rings of the chromophore coprecipitated with the DNA. Most of this fluorescence remained attached to DNA through successive reprecipitations, suggesting formation of covalent adducts between chromophore and DNA. Enzymatically digested poly(dA-dT) . poly(dA-dT)-chromophore adduct contained, in addition to deoxyadenosine and thymidine, several highly fluorescent hydrophobic products, separable by reverse-phase chromatography, all of which contained both adenine and thymine radiolabel, as well as chromophore radiolabel. One such product consistently had twice as much thymine as adenine, suggesting a structure chromophore-d(TpApT), in which the attached chromophore rendered both phosphodiester bonds refractory to endonuclease S1. This adduct fragment was completely hydrolyzed at pH 12, releasing adenine, 3'-dTMP, and 5'-dTMP. At pH 7, the adduct fragment slowly released chromophore and 3'-dTMP with parallel kinetics, leaving a modified d(ApT), which was cleaved by snake venom phosphodiesterase to yield 5'-dTMP and a modified deoxyadenosine. These hydrolysis patterns are unlike those of any previously characterized base or phosphotriester DNA adduct but rather indicate an altered deoxyadenosine sugar. The formation of adducts containing a modified deoxyribose suggests that deoxyribose may be the site of covalent chromophore attachment. Alteration of this same site, possibly the 5'-carbon of the sugar moiety, may account for the extreme lability of the phosphodiester bond.
Laser-stimulated fluorescence of submicrometer regions within single mitochondria of rhodamine-treated myocardial cells in culture.
Authors: Siemens A , Walter R , Liaw L H , Berns M W
Published: May 2006

A focused laser beam of 442 nm was used to stimulate fluorescence in 0.5-micrometer spots in single mitochondria of myocardial and endothelial cells in culture. Cells were treated with rhodamine 6G or 123 in order to render the mitochondria fluorescent. Rhodamine 123-treated cells exhibited a gradual decrease in fluorescence over several minutes, whereas the rhodamine 6G-treated myocardial cells exhibited three distinct patterns of variable fluorescence intensity. These patterns were detected at different points within the same mitochondrion or in different mitochondria. Mitochondria from nonmyocardial endothelial cells did not exhibit any variable intensity patterns of fluorescence. Electron microscopy revealed no ultrastructural damage attributable to laser exposure of the mitochondria. The variable fluorescence patterns are hypothesized to be indicative of localized alterations in molecules or ions at the suborganelle level.
DNA sequences of the joining regions of mouse lambda light chain immunoglobulin genes.
Authors: Blomberg B , Tonegawa S
Published: May 2006

The joining (J) segments of mouse immunoglobulin lambda light chain genes, lambda 2, lambda 3, and a presumptive lambda 4, were cloned, and their sequences were determined and compared with that of lambda 1. Although all the lambda J segments share sequence homology, the J1 and J4 segments and the J2 and J3 segments, respectively, are more homologous. These sequence data, together with the fact that present day lambda genes occur in two clusters, 5' J3C3J1C1 3' and 5' J2C2J4C4 3', further substantiates a probable evolutionary duplication unit, JIICIIJICI, with II the precursor of lambda 3 and lambda 2 and I the precursor of lambda I and lambda 4. From the J4 sequence, we conclude that the lambda 4 gene is most likely nonfunctional (i.e., a pseudogene). The signal nonamer sequence 5' to J3 differs from that of J1 in two consecutive base pairs. This difference could account in part for the lower level of expression of lambda 3 as compared with lambda 1 in mouse serum.
Specific-locus mutation frequencies in mouse stem-cell spermatogonia at very low radiation dose rates.
Authors: Russell W L , Kelly E M
Published: May 2006

Experiments were undertaken to augment the information on the lowest radiation dose rates feasible for scoring transmitted induced mutations detected by the specific-locus method in the mouse. This is the type of information most suitable for estimating genetic hazards of radiation in man. The results also aid in resolving conflicting possibilities about the relationship between mutation frequency and radiation dose rate at low dose rates. There was no statistically significant difference between mutation frequencies obtained in spermatogonia with 300 R (1 R = 2.6 x 10(-4) coulombs/kg) of gamma radiation at two different dose rates, 0.005 and 0.0007 R/min, or between either of these frequencies and data obtained earlier at dose rates of 0.8 R/min and below. This supports the view in an earlier publication by one of us (W.L.R.) that, at approximately 0.8 R/min and below, mutation frequency is independent of dose rate. Because this independence is now shown to extend over the more than 1000-fold range from 0.8 to 0.0007 R/min, it seems likely that it would hold at still lower dose rates, perhaps even to the much lower dose rates encountered in most human exposures.
cDNA clones coding for the heavy chain of human HLA-DR antigen.
Authors: Lee J S , Trowsdale J , Bodmer W F
Published: May 2006

Two cDNA clones, pDRH1 and pDRH2, containing sequences specific for human HLA-DR antigens were isolated from a bank of cDNA clones made from partially purified HLA-DR mRNA from the human lymphoblastoid cell line Maja. The clones were specific for the Mr 34,000 HLA-DR antigen glycoprotein chain. The identity of these clones was established by (i) their ability to hybridize specifically to HLA-DR mRNA in a positive selection assay; (ii) mRNA species hybridizing to the cDNA clones were expressed in B-cell but not in T-cell or fibroblast cell cultures; and (iii) a nucleotide sequence in the longer clone, pDRH2, could be translated into an amino acid sequence that is identical to the limited NH2-terminal amino acid sequence available for the purified HLA-DR antigen Mr 34,000 chain. Analysis of DNA from human, mouse, and human--mouse somatic cell hybrid lines by Southern transfer of restriction endonuclease digests indicated that the HLA-DR heavy chain is encoded in chromosome 6. This finding is compatible with the location of at least one of the HLA-D/DR heavy chain genes within the HLA region. In addition, the sequences coding for HLA-DR heavy chain appear to be present in only one or a few copies in the genome and to be relatively simple in structure.
Relative equilibria of vortices in two dimensions
Authors: Palmore Julian I.
Published: May 2006

An old problem of the evolution of finitely many interacting point vortices in the plane is shown to be amenable to investigation by critical point theory in a way that is identical to the study of the planar
n
-body problem of celestial mechanics. For any choice of positive circulations of the vortices it is shown by critical point theory applied to Kirchhoff's function that there are many relative equilibria configurations. Each of these configurations gives rise to a stationary configuration of the vortices in a suitably chosen rotating coordinate system. A sharp lower bound on the number of stationary vortex configurations for the problem of point vortices interacting in the plane is given. The problem of point vortices in a circular disk is defined and it is shown that these estimates hold for stationary configurations of small size.
Heat shock and recovery are mediated by different translational mechanisms.
Authors: DiDomenico B J , Bugaisky G E , Lindquist S
Published: May 2006

When Drosophila cells are shifted from 25 degrees C to 37 degrees C, protein synthesis is rapidly redirected from the complex pattern characteristic of normal growth to the simple pattern of heat shock proteins (HSPs). On return to 25 degrees C, synthesis of normal proteins is gradually reactivated and that of HSPs is repressed. In quantifying many different recovery experiments, we found that preexisting mRNAs always behaved as a cohort, with messages for different proteins returning to translation at the same rate. Heat shock mRNAs (HS mRNAs), on the other hand, never behaved as a cohort. Their repression was asynchronous, with translation of hsp70 always the first and translation of hsp82 always the last to be repressed. Although recovery times varied enormously (depending on the severity of the heat treatment), repression of hsp70 was always correlated with restoration of normal synthesis, suggesting a link between the two events, hsp70 repression was not simply due to competition with reactivated 25 degrees C mRNAs. A general decline in the translation efficiency of hsp70 mRNA was not observed. Instead, an increasing number of messages were translationally inactivated, while those remaining in the translational pool retained full ribosome loading. Unlike inactive 25 degrees C mRNAs, which are stable during heat shock, inactive HSP mRNAs are degraded during recovery.
X-ray absorption spectroscopy of the dimeric iron site in azidomethemerythrin from Phascolopsis gouldii.
Authors: Hendrickson W A , Co M S , Smith J L , Hodgson K O , Klippenstein G L
Published: May 2006

X-ray absorption spectroscopy has been used to study the dimeric iron center in azidomethemerythrin from Phascolopsis gouldii. Absorption edge data confirm that the two iron atoms are present as Fe(III) and suggest a hexa-coordination site for each of the iron atoms. The extended x-ray absorption fine structure (EXAFS) analysis provides direct structural evidence of a mu-oxo bridge between the two iron atoms at an average Fe-O distance of 1.71-1.76 A. Analysis using a multiple-scattering formalism calculates upper limits of 165 degrees for the Fe-O-Fe bridging angle and 3.38 A for the Fe-Fe distance. This result agrees with current crystallographic models being determined by refinement of structures of two azidomethemerythrins.
Identification of a pterin as the acrasin of the cellular slime mold Dictyostelium lacteum.
Authors: Van Haastert P J , De Wit R J , Grijpma Y , Konijn T M
Published: May 2006

Cell aggregation in Dictyostelium discoideum is mediated by chemotaxis to cyclic AMP. Aggregative cells of the simpler species D. lacteum are not attracted by this cyclic nucleotide. We describe how the cell aggregation-inducing factor, or acrasin, of D. lacteum was purified from aggregating amoebae and characterized. The acrasin, which is mainly secreted in the aggregative phase, is identified as a derivative of pterin. This identification is based on (i) its UV spectrum, (ii) the inhibition of the enzymatic degradation of acrasin by 6-methylpterin, (iii) the antagonistic effect of 6-aminopterin on chemotaxis towards both pterin and acrasin and not on the response towards folic acid or cyclic AMP, and (iv) the degradation of the acrasin to pterin. Its chromatographic properties show that the acrasin is an as yet unidentified pterin derivative. The acrasin is species specific and attracts cells at very low concentrations (0.1-0.01 microM). Also, several naturally occurring stereoisomers of 6-polyhydroxyalkylpterins attract aggregative cells at these low concentrations. Additionally, we identified a pterin deaminase, which converts pterin into 2-deamino-2-hydroxypterin (lumazin), as the acrasinase in D. lacteum.
Rhodopsin-to-metarhodopsin II transition triggers amplified changes in cytosol ATP and ADP in intact retinal rod outer segments.
Authors: Zuckerman R , Schmidt G J , Dacko S M
Published: May 2006

We have observed rapid, light-initiated changes in unbound cytosol ATP and ADP during the rhodopsin-to-metarhodopsin II transition in intact rod outer segments (ROS). Upon illumination of the ROS, ATP is rapidly removed from the unbound phase of ROS, accompanied by the concomitant release of ADP into the cytosol. The exchange process involves decreases of approximately equal to 0.5 mM ATP in ROS cytosol ATP content in response to a saturating flash. At levels of light well below saturation (less than 0.001% bleach), the process is highly amplified, with a decrease in cytosol ATP of approximately equal to 2,000 ATP molecules per absorbed photon per ROS. Rapid time-resolution techniques reveal that cytosol ATP content decreases rapidly, within 250 msec of a saturating flash. Bleaching rhodopsin to metarhodopsin II results in a decrease in cytosol ATP, accompanied by an increase in cytosol ADP, whereas photoreversal of metarhodopsin II by a blue flash reverses the process, increasing ATP concentration to its control level in the dark. The photoreversibility of the ATP decrease during the rhodopsin-to-metarhodopsin II transition establishes a direct link between the state of an early intermediate of photolyzed rhodopsin and the state of a nucleoside triphosphate in intact ROS. The results are consistent with a light-activated exchange of unbound ATP for bound ADP, and we propose, therefore, an ATP/ADP amplification cycle in which metarhodopsin II catalyzes the exchange of ATP for ADP on a nucleotide binding protein.
Amino acid sequence of a basic blue protein from cucumber seedlings
Authors: Murata M. , Begg G. S. , Lambrou F. , Leslie B. , Simpson R. J. , Freeman H. C. , Morgan F. J.
Published: May 2006

The amino acid sequence of a type 1 copper protein, the 96-residue basic blue protein from cucumber seedlings, has been determined by Edman degradation of the intact molecule and of fragments produced by cleavage with cyanogen bromide and with trypsin. The cucumber basic blue protein shows a marked sequence homology with stellacyanin, and to a smaller degree with plastocyanin and azurin. The known copper ligands of plastocyanin and azurin (corresponding to histidine-37, cysteine-84, histidine-87, and methionine-92 in plastocyanin) are present in the cucumber basic blue protein. However, the latter also contains a half-cystine residue analogous to the suggested fourth ligand of stellacyanin, where methionine is absent.
Evolution of tumor cell heterogeneity during progressive growth of individual lung metastases.
Authors: Poste G , Tzeng J , Doll J , Greig R , Rieman D , Zeidman I
Published: May 2006

The metastatic properties of tumor cell clones isolated from individual lesions of B16 melanoma metastatic to lung have been examined at different stages in the evolution of metastasis. Clonal analysis of metastatic lesions produced by B16 melanoma populations containing clones with identifiable, stable drug-resistance markers revealed that the majority (greater than 80%) of experimental metastases produced by intravenous injection of tumor cells are of unicellular origin. During the early stages of their growth (less than 25 days after initial tumor cell arrest), the majority of metastatic lesions contain cells with indistinguishable metastatic phenotypes (intralesional clonal homogeneity) although different clonally homogeneous lesions from the same host contain tumor cells with different metastatic phenotypes (interlesional clonal heterogeneity). Progressive growth of metastatic lesions is accompanied by emergence, within originally clonally homogeneous lesions, of variant tumor cells with altered metastatic properties (intralesional clonal heterogeneity). By 40-45 days after initial arrest of injected tumor cells in the lung, 90% of the metastatic lesions are populated by cells with heterogeneous metastatic phenotypes.
Expression of herpes simplex virus glycoprotein C from a DNA fragment inserted into the thymidine kinase gene of this virus.
Authors: Lee G T , Pogue-Geile K L , Pereira L , Spear P G
Published: May 2006

Previous reports have described mutants of herpes simplex virus type 1 that fail to produce or accumulate one of the major glycoproteins, glycoprotein C (gC). This defect is not lethal in cell culture, has been associated with the syncytial plaque morphology of some mutants, and may result from mutations that map to a region on the genome noncontiguous with the structural gene for gC. To investigate the conditions required for, and consequences of, gC expression in a specific genetic background, we have inserted a wild-type allele of the gC gene into the thymidine kinase gene (tk) of a gC- fusion-inducing viral mutant, strain MP. This was accomplished by identifying cloned viral DNA fragments homologous to gC mRNA, inserting the appropriate fragments into the viral tk cloned in pBR322, and then cotransfecting cells with the recombinant plasmids and DNA from strain MP, for selection of insertional TK- mutants. All TK- mutants containing insertions of appropriate sequences (in either orientation) into tk were found to express gC while maintaining the syncytial plaque morphology of strain MP. Elimination of the insertion from one of the TK- mutants was accompanied by loss of ability to produce gC. Our results permit more precise mapping of the DNA sequence encoding gC, to a subfragment of Sal I fragment R (map coordinates 0.620-0.640) and indicate also that promoter sequences for the gC gene may be located in this fragment. Moreover, we can conclude that the previously described regulatory mutation of strain MP does not prevent expression of gC from the DNA inserted into its gene tk and that the syncytial phenotype of MP cannot be due solely to absence of gC.
Conserved regions of homologous G-banded chromosomes between orders in mammalian evolution: carnivores and primates.
Authors: Nash W G , O'Brien S J
Published: May 2006

The recent derivation of a biochemical map of 33 loci of the domestic cat (Felis catus) revealed a striking conservation of chromosomal linkage associations between the cat and humans. A comparison of homologous (by linkage criteria) chromosomes by using conventionally extended and high-resolution G-banding of human and feline chromosomes is presented. Four criteria for establishing probable cytogenetic homologies of chromosomal regions were invoked: (i) map placement of homologous genes to the same chromosomes; (ii) cytological correlation of G-banding pattern; (iii) placement of homologous genes, by regional gene mapping, in the region of cytological homology; and (iv) a requirement that the putative region of homology be ancestral and evolutionarily conserved within their respective orders. Five subchromosomal regions (homologous to human chromosome 1p, 2p, 2q, 12, and X) were found to be conserved and homologous by all the stated criteria. The conserved regions constitute nearly 20% by length of the human chromosomal genome. The implications of conservation of chromosome homologies between mammalian orders whose last common ancestor became extinct more than 60 million years ago is discussed.
A phylogeny for the principal alleles of the human phosphoglucomutase-1 locus.
Authors: Takahashi N , Neel J V , Satoh C , Nishizaki J , Masunari N
Published: May 2006

The results of phosphoglucomutase-1 (PGM1) typings by starch gel electrophoresis and subtypings by isoelectric focusing are presented for a sample of Japanese. A distinction made on the basis of isoelectric focusing (termed "+" and "-") is nonrandomly associated with each of the products of the four most common electrophoretic alleles (PGM1(1), PGM1(2), PGM1(3), and PGM1(7). The isoelectric trait cosegregates with the allele; the degree of nonrandomness of the association varies from allele to allele. Thus, the four alleles become eight. On the basis of these facts plus the additive nature of the pI differences between allele products and the geographical distribution of the alleles, an allele phylogeny can be constructed. This postulates that the eight alleles may be explained by three nucleotide substitutions involving the stem allele plus four intragenic recombinations between these substitutions. The potential of intragenic recombination as a cause of mutation has been insufficiently appreciated.
Antigen-specific human T-cell hybridomas with helper activity.
Authors: DeFreitas E C , Vella S , Linnenbach A , Zmijewski C , Koprowski H , Croce C M
Published: May 2006

Human T cell hybridomas were produced by fusing the hypoxanthine phosphoribosyltransferase-deficient line of the human T cell lymphoma Jurkat with a continuous line of normal human T cells specific for tetanus toxoid (TeT). The hybridomas were selected for their ability to produce interleukin 2 after exposure to TeT on semiautologous monocytes and for their ability to bind to TeT-pulsed semiautologous monocytes. These antigen-specific T hybridomas demonstrated potent helper activity for semiautologous B cells as determined by the production of high levels of anti-TeT antibody in vitro.
Human hybridomas constructed with antigen-specific Epstein-Barr virus-transformed cell lines.
Authors: Kozbor D , Lagarde A E , Roder J C
Published: May 2006

A 6-thioguanine-resistant, human lymphoblastoid B-cell line (GM1500 6TG A-11; IgG secreting) was mutagen-treated with low-level gamma-irradiation and selected for ouabain resistance. One line showing 10,000-fold higher drug resistance, designated KR-4, was fused with an Epstein-Barr virus-transformed, cloned, B-lymphocyte cell line (B6) producing antitetanus toxoid (TT) antibody (IgM), and the hybrids were selected in hypoxanthine/aminopterin/thymidine medium containing 10 microM ouabain. Surviving cells, which arose at an optimal frequency of 10(-5), were subcloned by limiting dilution and screened for anti-TT production. Out of 395 final subclones, 372 were found positive for anti-TT, and seven that were selected for further study secreted specific antibody (IgM, kappa chain) at a maximum concentration of 3-6 micrograms/ml. The differential rate of anti-TT production during the logarithmic phase of cell growth was 15-fold higher in the hybridomas than in the original B6 line. The hybrid nature of the clones was confirmed by karyotype analysis, histocompatibility antigen typing, and expression of secreted and membrane-bound Ig classes. Biosynthetic labeling of the cells revealed that all hybrids secreted both IgM and IgG but that only the IgM class had specificity for TT. Because Epstein-Barr virus is a polyclonal B-lymphocyte activator, the technique we applied here may be useful for increasing the recovery of rare antigen-specific B cells in the peripheral blood and for improving the frequency and stability of hybridomas secreting a given antibody.
Regulation of intracellular calcium compartmentation: studies with isolated hepatocytes and t-butyl hydroperoxide.
Authors: Bellomo G , Jewell S A , Thor H , Orrenius S
Published: May 2006

In suspensions of isolated hepatocytes, two intracellular Ca2+ pools were distinguished in the presence of the metallochrome indicator arsenazo III, first by treatment with the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and then with the Ca2+ ionophore A23187. The available evidence indicates that the two pools are of mitochondrial and extramitochondrial origin. Metabolism of t-butyl hydroperoxide by hepatocytes caused release of Ca2+ from both compartments concomitant with oxidation of cellular glutathione and NADPH, which was followed by characteristic alterations in cell surface structure. When NADPH oxidation was prevented by selective inactivation of glutathione reductase, t-butyl hydroperoxide metabolism was without effect on the mitochondrial Ca2+ pool, whereas the loss from the extramitochondrial pool was accelerated. Our results suggest that different regulatory mechanisms modulate mitochondrial (NADPH-dependent) and extramitochondrial (thiol-dependent) Ca2+ compartmentation and that disturbance of normal Ca2+ homeostasis may be critical in peroxide-induced cytotoxicity.
Human leukocyte elastase, cathepsin G, and lactoferrin: family of neutrophil granule glycoproteins that bind to an alveolar macrophage receptor.
Authors: Campbell E J
Published: May 2006

Interactions between polymorphonuclear neutrophils and mononuclear phagocytes are potentially of great importance in a variety of inflammatory processes. As part of a continuing effort to elucidate the physiologic importance of human alveolar macrophage receptor-mediated binding of neutrophil (leukocyte) elastase, I have studied the binding of leukocyte elastase and two other neutrophil granule glycoproteins, cathepsin G and lactoferrin, to human alveolar macrophages. Saturable binding of all three ligands at 0 degrees C was observed, with equilibrium dissociation constants of 4.0 x 10(-7), 2.0 x 10(-7), and 1.7 x 10(-6) M, respectively. All bound to a similar number (54-73 x 10(6)) of sites per cell. Binding of all three ligands was inhibited by the polysaccharide fucoidin, and extensive cross-inhibition of their binding to macrophages was observed. The results indicate that alveolar macrophages possess a relatively low-affinity, high-volume receptor for a family of neutrophil granule glycoproteins, which would be ideally suited for clearing released neutrophil granule contents from the extracellular space in inflamed tissues.
Induction of pole cells in sterilized
Drosophila
embryos by injection of subcellular fraction from eggs
Authors: Ueda Ryu , Okada Masukichi
Published: May 2006

A subcellular fraction isolated from a homogenate of young
Drosophila
embryos was shown to be capable of inducing pole cells when injected into UV-sterilized
Drosophila
embryos. Most of the pole cell-inducing activity was recovered from the precipitate after centrifugation at 27,000 ×
g
. The activity remained in this precipitate (called F-3 fraction hereafter) even after membranous structures were removed from it through centrifugation on a sucrose density gradient. Dialysis, lyophilization, and heating at 80°C for 10 min did not inactivate the F-3 fraction. The pole cells, which were produced when the F-3 fraction was injected at the posterior pole of UV-sterilized embryos, did not develop into germ cells. Furthermore, the F-3 fraction was unable to induce pole cells when injected into the anterior region of the egg. These results can be explained by assuming that (
i
) pole cell formation and germ cell determination are controlled by different factors, (
ii
) pole cell formation requires at least two factors, which are normally localized in the posterior-pole cytoplasm, one of which is sensitive and one resistant to the UV dosage we used, and (
iii
) the subcellular fraction we obtained contains the UV-sensitive factor but not the UV-resistant factor.
Selective amplification of polymorphic dihydrofolate reductase gene loci in Chinese hamster lung cells.
Authors: Lewis J A , Davide J P , Melera P W
Published: May 2006

A series of antifolate-resistant Chinese hamster lung sublines that overproduce either a Mr 20,000 or a Mr 21,000 class of dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3), known to contain amplified DHFR genes, has been analyzed by DNA and RNA transfer techniques. The results suggest that the Mr 20,000 and Mr 21,000 DHFRs are encoded by at least two polymorphic DHFR genes, both of which are expressed in drug-sensitive parental cells. In drug-resistant sublines only one of the two DHFR gene types is amplified, thus accounting for the overproduction of one or the other molecular weight class of DHFR. In addition to the known differences between the DHFRs whose overproduction they direct, these allelic genes differ in restriction endonuclease profiles and in the relative abundances of their multiple mRNA transcripts.
Isolation of cDNA clones encoding HLA-DR alpha chains.
Authors: Wake C T , Long E O , Strubin M , Gross N , Accolla R , Carrel S , Mach B
Published: May 2006

HLA-DR antigens, the human equivalent of mouse Ia antigens, are multimeric surface glycoproteins characterized by a high degree of allelic polymorphism. They are expressed specifically on macrophages and lymphocytes and they play a key role in the regulation of the immune response. We have investigated this complex genetic system by a direct study of the genes involved through molecular cloning. This paper deals with the cloning, in plasmids, of full-length cDNA sequences for the HLA-DR alpha chain from the human B-cell line Raji. The approach relies on a translation assay of mRNA injected into frog oocytes and recognition of translation products by polyclonal and monoclonal antibodies. After enrichment of specific mRNA and cloning of cDNA, plasmid clones were analyzed by hybridization-selection of mRNA and translation in oocytes. A clone was identified and used to screen a cDNA library from which several full-length HLA-DR alpha chain plasmids were isolated. DNA sequence determination of one such clone confirmed its identity and also established the amino acid sequence of the NH2-terminal signal sequence of HLA-DR alpha chains. The translation product of HLA-DR alpha chain mRNA purified by hybridization-selection gives a single alpha chain spot on two-dimensional gels, whereas the alpha chain released from the alpha/beta HLA-DR complex gives about seven distinct spots. Finally, the results of analysis of genomic DNA by Southern blotting are compatible with the existence of a single nonpolymorphic alpha chain gene and indicate extensive cross-hybridization with a homologous gene in mouse DNA.
Effects of skeletal muscle protein phosphatase inhibitor-2 on protein synthesis and protein phosphorylation in rabbit reticulocyte lysates
Authors: Ernst Vivian , Levin Daniel H. , Foulkes J. Gordon , London Irving M.
Published: May 2006

Reticulocyte lysates contain two major classes of protein phosphatase activities, designated type 1 and type 2. These designations are based on criteria derived from the analyses of protein phosphatase species in other tissues. The criteria include (
i
) chromatographic elution profiles on DEAE-cellulose; (
ii
) specificity of lysate phosphatases toward [
32
P]phosphorylase
a
and [
32
P]phosphorylase kinase; (
iii
) sensitivity of lysate phosphatases to Mg
2+
ATP; and (
iv
) sensitivity to the heat-stable protein phosphatase inhibitor-2. The lysate phosphatase species are similar to those described in rabbit skeletal muscle and rabbit liver. Reticulocyte protein phosphatase type 1, but not type 2, is inhibited by heat-stable protein phosphatase inhibitor-1 and -2 which have been characterized from rabbit skeletal muscle. We have initiated a study on the function and specificity of lysate protein phosphatase activities involved in the regulation of protein synthesis by examining the effects of protein phosphatase inhibitor-2 on reticulocyte protein synthesis and protein phosphorylation. Our findings are as follows. (
a
) Protein phosphatase inhibitor-2 inhibits protein chain initiation in hemin-supplemented lysates. (
b
) Inhibition is characterized by biphasic kinetics and is reversed by the delayed addition of purified reticulocyte eukaryotic initiation factor 2 (eIF-2). (
c
) Inhibition of protein synthesis by inhibitor-2 is accompanied by the phosphorylation of the α-subunit (38,000 daltons) of eIF-2 (eIF-2α) and of two heat-stable polypeptides of 29,000 and 44,000 daltons. (
d
) The 29,000-dalton component is phosphorylated in lysates under conditions of protein synthesis and appears to be inhibitor-2, but the physiological significance of this modification of inhibitor-2 is not clear. (
e
) Inhibitor-2 has no effect on the activation
in vitro
of isolated heme-regulated or double-stranded RNA-dependent eIF-2α kinases. We propose that the inhibition of protein synthesis in hemin-supplemented lysates by added inhibitor-2 is due at least in part to the inhibition of a type 1 eIF-2α phosphatase activity, which permits a basal eIF-2α kinase activity to be expressed leading to the accumulation of phosphorylated eIF-2α and an inhibition of protein synthesis.
Replacement of anticodon loop nucleotides to produce functional tRNAs: amber suppressors derived from yeast tRNAPhe.
Authors: Bruce A G , Atkins J F , Wills N , Uhlenbeck O , Gesteland R F
Published: May 2006

The method of anticodon loop replacement has been used to make derivatives of yeast tRNAPhe. By constructing tRNAs with a CUA anticodon, complementary to the amber (UAG) terminator, functional amber suppressor tRNAs were produced. The activity of these tRNAs was assayed in a mammalian cell-free protein synthesizing system. The level of suppression reflects the efficiency of codon recognition. tRNAs were constructed with either A, C, U, or G on the 3' side of the CUA anticodon. The tRNAs containing the purines were efficient amber suppressors, whereas those containing pyrimidines were inefficient.