bluuebunny/crossref_metadata_2025 huggingface dataset:
Denervated skeletal muscle displays discoordinate regulation for the synthesis of several myofibrillar proteins.
Authors: Matsuda R , Spector D , Strohman R C
Published: May 2006

Synthesis patterns of myosin heavy- and light-chain isoforms, tropomyosin and troponin, have been studied in chicken fast muscle denervated at both neonatal and adult stages. Denervated neonatal muscle does not synthesize the adult myosin heavy-chain isoform at the time of denervation, but it does synthesize the adult isoform several months after denervation. Thus, innervation does not appear to be necessary for the normal sequential replacement of embryonic and neonatal myosin heavy chain by the adult variant. Nerve is required, however, for the regulation of tropomyosin and troponin expression. Normally the pectoralis major muscle represses synthesis of both beta-tropomyosin and leg-type troponin T during late embryonic development. After denervation, however, the muscle relaxes its ongoing repression of these proteins and significant amounts of both beta-tropomyosin and leg-type troponin T are synthesized by the muscle. Denervation also results in an altered pattern of myosin light-chain synthesis so that the ratio of fast light-chain 3/fast light-chain 1 decreases. Similar results are found in muscle denervated at the adult stage. In denervated muscle, therefore, synthesis of these myofibrillar proteins is not coordinated: ongoing isoform shifts proceed to express an adult pattern of myosin heavy chain while tropomyosin, troponin, and myosin light-chain patterns appear to revert to embryonic configurations.
Ca2+-ATPase of the sarcoplasmic reticulum shares a common domain with a membrane glycoprotein associated with the cytoskeleton of microvilli.
Authors: Reggio H , Coudrier E , Tokuyasu K , Louvard D
Published: May 2006

On the basis of structural observations, it has been proposed that cytoskeletal organization of the intestinal microvilli could be related to striated muscle structure. We have prepared antibodies against an amphipathic membrane glycoprotein (140 kilodaltons) associated with microvillar cytoskeleton and investigated its occurrence in striated muscle. Frozen sections of striated muscle were prepared according to the technique of Tokuyasu and visualized by indirect immunofluorescence with antibodies to the 140-kilodalton protein. In longitudinal sections the labeling was concentrated mainly in the area of the I band. In cross sections a honeycomb pattern was observed, suggesting that the recognized antigen was probably associated with the periphery of the myofibrils. Ultrathin frozen sections prepared for electron microscopy revealed that this antigen is closely associated with the membrane of the sarcoplasmic reticulum. In muscle extracts, the antibodies to the intestinal microvillar 140-kilodalton protein recognized a protein of 100 kilodaltons that comigrates with the Ca2+-ATPase of the sarcoplasmic reticulum. They recognized a purified preparation of the Ca2+-ATPase and, more specifically, the trypsin-generated fragment A2, the NH2-terminal part of the molecule that is exposed on the cytoplasmic face of the sarcoplasmic reticulum. Although these two proteins, expressed in unrelated cells, have a different molecular size and are inserted in different types of membranes, they share a common structural domain responsible for their crossreactivity. We propose that this domain could also be responsible for a common function--namely, the bridging of actin filaments to membranes.
X chromosome-linked muscular dystrophy (mdx) in the mouse.
Authors: Bulfield G , Siller W G , Wight P A , Moore K J
Published: May 2006

An X chromosome-linked mouse mutant (gene symbol, mdx) has been found that has elevated plasma levels of muscle creatine kinase and pyruvate kinase and exhibits histological lesions characteristic of muscular dystrophy. The mutants show mild clinical symptoms and are viable and fertile. Linkage analysis with four X chromosome loci indicates that mdx maps in the Hq Bpa region of the mouse X chromosome. This gives a gene order of mdx-Tfm-Pgk-1-Ags, the same as for the equivalent genes on the human X chromosome.
Characteristics of human lipoproteins isolated by selected-affinity immunosorption of apolipoprotein A-I.
Authors: McVicar J P , Kunitake S T , Hamilton R L , Kane J P
Published: May 2006

We have isolated high density lipoproteins (HDL) from human serum by a new strategy, selected-affinity immunosorption, avoiding perturbation from ultracentrifugation or polyanion precipitation. The principle of this strategy is to utilize a subpopulation of monospecific antibodies directed against apolipoprotein A-I (apo A-I), which was preselected for dissociation under mild conditions of elution. Particles containing more than 90% of the apo A-I contained in serum were isolated uncontaminated by any other serum proteins. The immunoaffinity columns have been cycled over 300 times without apparent diminution in capacity. The apo A-I-containing particles sequestered from serum by immunosorption were more polydisperse in diameter and contained more protein than in ultracentrifugally isolated HDL. They also contained minor apolipoproteins that were not observed in ultracentrifuged HDL. Furthermore, the apo A-I-containing particles had different electrophoretic mobilities from those of ultracentrifugally isolated HDL when run under nondenaturing conditions in polyacrylamide gels. The apo A-I-containing particles isolated by our procedure separate into discrete bands similar to the subspecies visualized when whole serum is subjected to electrophoresis, whereas ultracentrifuged HDL migrate as two broad featureless zones. This suggests that selected affinity immunosorption does not subject the lipoproteins to structural disruption like that which occurs during ultracentrifugation. The principle of selected-affinity immunosorption should be of widespread utility in the isolation of fragile biological complexes.
Increased rates of polypeptide chain elongation in placental explants from human diabetics.
Authors: Ilan J , Pierce D R , Hochberg A A , Folman R , Gyves M T
Published: May 2006

Average rates of polypeptide chain elongation were determined in placental explants of first trimester and term placentas from both normal and diabetic human pregnancies. Average ribosome half-transit times were determined by measuring the kinetics of transfer of labeled polypeptides from polysomal-bound to released polypeptides. The average half-transit time decreases from 75 sec per ribosome in first trimester explants to 56 sec per ribosome in term placentas. The average polypeptide molecular weights synthesized by explants from first trimester and in term are 49,300 and 49,600, respectively, which are not significantly different. The average elongation rates for first trimester and term placental explants are 172 and 231 amino acids per minute per ribosome, respectively, which are significantly different. Moreover, the average polypeptide molecular weight synthesized by term placentas from diabetic pregnancies is 48,200, while the average ribosome half-transit time is 40 sec. Thus, ribosomes from explants of term placenta from diabetics move along the average message at a much higher speed than do ribosomes in normal term tissue. The assembly rate of amino acid into polypeptide in explant of placenta of diabetic mothers is 314 amino acids per minute, which is significantly faster than 231 amino acids per minute in normal term tissue. These findings indicate that during placental development and in diabetic pregnancy there is a large change in the actual rates at which amino acids are added to the nascent polypeptide chain--i.e., the rates in polypeptide chain elongation. Therefore, translation-level regulation of protein synthesis in placenta plays a significant part in the magnitude of the response to developmental and other physiological stimulations.
Resistance of herpes simplex virus to 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine: physical mapping of drug synergism within the viral DNA polymerase locus.
Authors: Crumpacker C S , Kowalsky P N , Oliver S A , Schnipper L E , Field A K
Published: May 2006

A herpes simplex virus type 2 (HSV-2) mutant TS6 (strain HG52) induces a heat-labile viral DNA polymerase at the nonpermissive temperature and is markedly resistant to 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]-guanine [2'-nor-2'-deoxyguanosine; 2'NDG]. This antiviral drug requires HSV thymidine kinase for phosphorylation to an active inhibitor (2'NDG-triphosphate), and thymidine kinase-deficient mutants of HSV exhibit varying degrees of resistance to 2'NDG, with the HSV type 1 (HSV-1) B2006 mutant (Kit) being markedly resistant. The ts6 mutation and the 2'ndgR-1 mutation within the viral DNA polymerase locus have been physically mapped by marker rescue and generation of HSV-1/HSV-2 intertypic recombinants. The physical map limits for the ts6 mutation and 2'ndgR-1 mutation are closely linked within a 2.2-kilobase-pair region of DNA sequences and are physically separate from the paaR-1 and acvR-1 mutations. Resistance to 2'NDG by HSV-2 ts6 can be overcome in the presence of combinations of 2'NDG and phosphonoacetic acid, indicating drug synergism within the viral DNA polymerase locus. These physical mapping studies expand the limits of DNA sequences defining an active center in the viral polymerase to 3.5 kilobase pairs, indicating that regions spanning the entire polymerase polypeptide may contribute to a specialized surface able to interact with nucleotides of different structure.
Diffusion and binding constants for acetylcholine derived from the falling phase of miniature endplate currents.
Authors: Land B R , Harris W V , Salpeter E E , Salpeter M M
Published: May 2006

In previous papers we studied the rising phase of a miniature endplate current (MEPC) to derive diffusion and forward rate constants controlling acetylcholine (AcCho) in the intact neuromuscular junction. The present study derives similar values (but with smaller error ranges) for these constants by including experimental results from the falling phase of the MEPC. We find diffusion to be 4 X 10(-6) cm2 s-1, slightly slower than free diffusion, forward binding to be 3.3 X 10(7) M-1 s-1, and the distance from an average release site to the nearest exit from the cleft to be 1.6 micron. We also estimate the back reaction rates. From our values we can accurately describe the shape of MEPCs under different conditions of receptor and esterase concentration. Since we suggest that unbinding is slower than isomerization, we further predict that there should be several short "closing flickers" during the total open time for an AcCho-ligated receptor channel.
Monoclonal antibodies against the rat liver glucocorticoid receptor.
Authors: Okret S , Wikström A C , Wrange O , Andersson B , Gustafsson J A
Published: May 2006

Splenic cells from one BALB/c mouse and one C57/BL mouse, immunized with purified rat liver glucocorticoid receptor (GR), were fused with the mouse myeloma cell line Sp 2/0-Ag 14. Screening for production of anti-GR-antibodies by the hybridomas was carried out with an enzyme-linked immunosorbent assay, using partially purified rat liver GR as antigen. Further screening was by a second-antibody immunoprecipitation assay using [3H]triamcinolone acetonide-GR complex from rat liver cytosol as tracer. Hybridomas from 10 different microplate wells, positive in both assays, were successfully cloned by the limiting dilution method to monoclonality. The different origins of the monoclonal antibodies were confirmed by their various isoelectric points when analyzed by isoelectric focusing. Four of the monoclonal hybridoma cell lines secreted IgM antibodies; two, IgG1; three, IgG2a; and one, IgG2b. The GR-antibody complex was identified in glycerol density gradients by a shift of the 4S GR to an 8.5S or 19S GR-antibody complex when incubated with monoclonal IgG or IgM antibody, respectively. The 10 monoclonal antibodies recognized different determinants on the GR, all situated on that domain of the receptor that is separate from the ligand and DNA-binding domains. Also, the cross-reactivity to the mouse liver GR varied among the monoclonal antibodies. No cross-reactivity was observed to the human lymphocytic GR. NaDodSO4 electrophoresis of a 0.5% pure GR preparation followed by immunoblotting using one of the monoclonal antibodies identified a single peptide with a molecular weight of 94,000, identical to the purified rat liver GR.
Purification in a functional form of the terminal protein of Bacillus subtilis phage phi 29.
Authors: Prieto I , Lázaro J M , García J A , Hermoso J M , Salas M
Published: May 2006

Phage phi 29 terminal protein, p3, essentially pure, was isolated in a denatured form from viral particles, and anti-p3 antiserum was obtained. A radioimmunoassay to detect and quantitate protein p3 was developed. By using this assay, native protein p3 was highly purified from Escherichia coli cells harboring a gene 3-containing recombinant plasmid. After three purification steps, the protein was more than 96% pure; its amino acid composition was very similar to that deduced from the nucleotide sequence of gene 3. The purified protein was active in the formation of the covalent p3-dAMP initiation complex when supplemented with extracts of B. subtilis infected with a sus mutant of phi 29 in gene 3. No DNA polymerase or ATPase activities were present in the final preparation of protein p3.
A quantitative dot-immunobinding assay for proteins using nitrocellulose membrane filters.
Authors: Jahn R , Schiebler W , Greengard P
Published: May 2006

An immunoassay method is described for the quantitative determination of synapsin I (protein I) and of a 36,000-dalton membrane protein from rat brain synaptic vesicles. The samples are spotted on nitrocellulose membrane filters, incubated sequentially with specific antibodies and 125I-labeled protein A, and assayed for radioactivity in a gamma scintillation counter. Conditions have been established to prevent losses of protein from the sheets during processing, to quench background radioactivity, and to adjust the sensitivity to the range desired. A large number of samples can be handled in parallel. The assay does not require iodination of the antigen and is accurate even with crude tissue samples. Standard curves were linear over a 20- to 50-fold range. The sensitivity of the method is such that 10 pmol of synapsin I and 50 ng of total vesicle membrane protein could be measured with accuracy. The method should prove useful for a wide range of proteins.
O4-ethyldeoxythymidine, but not O6-ethyldeoxyguanosine, accumulates in hepatocyte DNA of rats exposed continuously to diethylnitrosamine.
Authors: Swenberg J A , Dyroff M C , Bedell M A , Popp J A , Huh N , Kirstein U , Rajewsky M F
Published: May 2006

In previous investigations into the mechanisms responsible for cell specificity in hepatocarcinogenesis, we have demonstrated that O6-methylguanine accumulates in the DNA of nonparenchymal cells (NPC) but is efficiently removed from hepatocellular DNA. O6-Alkylguanine may, therefore, be an important promutagenic lesion responsible for the induction of hepatic angiosarcomas after exposure to methylating agents, but other promutagenic DNA alkylation products--i.e., O4-alkylthymine--may be responsible for the initiation of hepatocellular carcinomas. F-344 male rats were provided drinking water containing diethylnitrosamine (DEN) at 40 ppm for 0, 2, 4, 8, 16, 28, 49, or 77 days, a regimen that selectively causes hepatocellular carcinomas. Hepatocytes and NPC were isolated by using low-speed differential centrifugation. DNA was purified by hydroxyapatite chromatography and hydrolyzed enzymatically, and O4-ethyldeoxythymidine (O4-EtdThd) and O6-ethyldeoxyguanosine (O6-EtdGuo) of hepatocyte and NPC DNA were quantitated by competitive radioimmunoassay using high-affinity monoclonal antibodies. O4-EtdThd accumulated in hepatocyte DNA during the first 28 days of DEN exposure, approximating a steady state at an O4-EtdThd-to-deoxythymidine molar ratio of approximately equal to 1 X 10(-5). This O4-EtdThd concentration was maintained from 28 to 77 days of DEN exposure. In contrast, O6-EtdGuo did not accumulate in hepatocyte DNA, its greatest concentration O6-EtdGuo-to-deoxyguanosine ratio (approximately equal to 3.7 X 10(-7) ) being detected after 2 days of exposure to DEN. O6-EtdGuo concentrations in hepatocyte DNA decreased with duration of exposure to DEN to an O6-EtdGuo-to-deoxyguanosine ratio of less than 2 X 10(-7) from 28 to 77 days. The data indicate that O4-EtdThd disappears from the DNA of hepatocytes less than 1/200th as fast as O6-EtdGuo. DNA from NPC contained approximately half as much O4-EtdThd as hepatocytes did, but greater than or equal to 2.5 times more O6-EtdGuo.
Monoclonal antibodies specific for a murine cytotoxic T-lymphocyte clone.
Authors: Staerz U D , Pasternack M S , Klein J R , Benedetto J D , Bevan M J
Published: May 2006

Two antibody-secreting murine hybridomas, F1G3.1 and F2A11.5, have been established from B10.D2 mice immunized with cells from the murine cytotoxic T-lymphocyte clone G4. The two clones used, G4 and B10, were derived from BALB.B (H-2b) mice and the target antigen specificity of both maps to the Dd region of the murine H-2 complex. However, B10 has a lower affinity for the target cells, as shown by its lower specific killing of blasts and its higher susceptibility to blocking by anti-Lyt-2 monoclonal antibody 53-6.75. The monoclonal antibodies, F1G3.1 and F2A11.5, react only with cells from clone G4. Similarly, they block only the specific cytolysis mediated by G4; no effect on cytotoxicity mediated by B10 or by heterogeneous populations of cytotoxic T lymphocytes was found. F1G3.1, especially, is very active in stimulating G4 to secrete immune interferon; B10 in contrast did not show any induction on treatment with these monoclonal antibodies. The structure of the surface antigen on G4 cells recognized by these monoclonal antibodies was revealed by immunoprecipitation studies of radioiodinated cell surface proteins. A protein dimer could be identified with an apparent molecular size of 80,000 daltons consisting of monomers migrating as 42,000-dalton proteins on reduction. So far, electrophoresis in the presence of NaDodSO4 does not indicate any heterogeneity in the size of the monomers. This molecule can be distinguished from the Lyt-2 complex.
Co-expression of corticotropin-releasing factor and vasopressin immunoreactivity in parvocellular neurosecretory neurons of the adrenalectomized rat.
Authors: Sawchenko P E , Swanson L W , Vale W W
Published: May 2006

To clarify the anatomical organization that allows for the synergy of vasopressin and oxytocin with corticotropin-releasing factor (CRF) in promoting adrenocorticotropic hormone secretion from the anterior pituitary, immunohistochemical double staining methods were used to compare the distribution of these peptides in the hypothalamic paraventricular nucleus of normal, colchicine-treated, and adrenalectomized male rats. In untreated animals, a few CRF-stained cells were found in the parvocellular division of the paraventricular nucleus, while brightly stained oxytocin- and vasopressin-immunoreactive cells were centered in the magnocellular division. In animals treated with colchicine, and inhibitor of axonal transport, large numbers of CRF-stained cells were found in the parvocellular division of the nucleus, and 1-2% of these also stained with antivasopressin. As reported previously, a substantial number of oxytocin-stained cells, centered in a discrete anterior part of the magnocellular division, also expressed CRF immunoreactivity. In contrast, after adrenalectomy, CRF immunostaining of cells in the parvocellular division was enhanced selectively and greater than 70% of these cells also stained positively for vasopressin. The distribution of oxytocin-stained cells was not influenced by adrenalectomy. The unusual localization of vasopressin immunoreactivity in parvocellular neurosecretory neurons in the adrenalectomized rat suggests that a single population of cells can produce CRF and vasopressin, both of which are potent promoters of adrenocorticotropic hormone secretion. These findings indicate that there is a state-dependent plasticity in the expression of biologically active peptides by individual neuroendocrine neurons.
Structure of human hemopexin: O-glycosyl and N-glycosyl sites and unusual clustering of tryptophan residues.
Authors: Takahashi N , Takahashi Y , Putnam F W
Published: May 2006

The primary structure of human hemopexin is being deduced from sequence analysis of a series of peptides obtained from chemical and enzymatic digests of the protein. Human hemopexin consists of about 440 amino acid residues. It has five sites of attachment of glucosamine oligosaccharides at the signal sequence of Asn-X-Thr/Ser. A unique structural feature is the virtual blocking of the amino-terminal threonine residue, which is O-linked to a galactosamine oligosaccharide that has not previously been identified in this protein. The galactosamine oligosaccharide and one glucosamine oligosaccharide are located in the amino-terminal region, three of the glucosamine oligosaccharides are in the middle region, and one glucosamine oligosaccharide is in the carboxyl-terminal region of the protein. Two of the five glucosamine oligosaccharides are present in a histidine-rich sequence of the middle region of the protein, in which the histidines flank beta-turns presumably at the surface of hemopexin. Clusters of tryptophan residues occur in four regions, each of which contains three or four tryptophan residues separated by 0-12 other residues. This clustering is significant because both histidine and tryptophan have been implicated in the binding of heme. A computer analysis did not identify significant matches of human hemopexin to any protein, including cytochromes and other heme-binding proteins, which suggests that the human hemopexin gene evolved from a unique primordial gene differing from those of other heme-binding proteins.
cAMP pulses coordinate morphogenetic movement during fruiting body formation of
Dictyostelium minutum
Authors: Schaap Pauline , Konijn Theo M. , van Haastert Peter J. M.
Published: May 2006

Aggregation in the primitive cellular slime mold
Dictyostelium minutum
proceeds by means of chemotaxis toward a continuously secreted folic acid analog [De Wit, R. J. W. & Konijn, T. M. (1983)
Cell Differ.
12, 205-210]. The onset of culmination is marked by the appearance of concentric waves of cell movement on the aggregate surface. Culmination proceeds by the chemotactic attraction of amoebae to the center of wave propagation, which results in the accumulation of amoebae into a finger-like structure. Evidence is presented that the chemoattractant used during culmination is cAMP, which is secreted in pulses. The cells secrete cAMP themselves; cAMP receptors and phosphodiesterase activity appear on the cell surface just before the onset of culmination. Micromolar concentrations of externally applied cAMP induce disorientation of amoeboid movement at the onset of culmination. These observations are compatible with the hypothesis that the cAMP signaling system organizes multicellular development in both primitive and advanced cellular slime mold species. Advanced species such as
Dictyostelium discoideum
use this signaling system also in an earlier stage of development to organize the process of cell aggregation.
Processing of lysozyme by macrophages: identification of the determinant recognized by two T-cell hybridomas.
Authors: Allen P M , Strydom D J , Unanue E R
Published: May 2006

The purpose of this study was to identify the fragment of the hen egg-white lysozyme (HEL) molecule presented by macrophages to helper T cells. This was investigated by using T-cell hybridomas and macrophages prefixed in paraformaldehyde. We previously had shown that such prefixed macrophages could present a tryptic digest of HEL. The tryptic peptides were separated by HPLC and tested for their ability to stimulate the T-cell hybridomas. Only one tryptic peptide was found to be immunogenic. This immunogenic peptide was identified as the tryptic peptide T-8, containing amino acids 46-61. The precise determinant on the peptide T-8 being recognized was further defined by testing the response of the two T-cell hybridomas to human lysozyme. Neither clone responded to human lysozyme. From the amino acid sequence of human lysozyme, the determinant was localized to the four amino-terminal residues. Cleavage of the immunogenic peptide with either chymotrypsin or protease V-8 completely abolished the immunogenicity. This suggested that the T-cell determinant is located in the hydrophilic amino-terminal residues and that it must be associated with a hydrophobic stretch of amino acids, which allows the peptide to associate with the macrophage plasma membrane.
Amino acid sequence of homologous rat atrial peptides: natriuretic activity of native and synthetic forms.
Authors: Seidah N G , Lazure C , Chrétien M , Thibault G , Garcia R , Cantin M , Genest J , Nutt R F , Brady S F , Lyle T A
Published: May 2006

A substance called atrial natriuretic factor (ANF), localized in secretory granules of atrial cardiocytes, was isolated as four homologous natriuretic peptides from homogenates of rat atria. The complete sequence of the longest form showed that it is composed of 33 amino acids. The three other shorter forms (2-33, 3-33, and 8-33) represent amino-terminally truncated versions of the 33 amino acid parent molecule as shown by analysis of sequence, amino acid composition, or both. The proposed primary structure agrees entirely with the amino acid composition and reveals no significant sequence homology with any known protein or segment of protein. The short form ANF-(8-33) was synthesized by a multi-fragment condensation approach and the synthetic product was shown to exhibit specific activity comparable to that of the natural ANF-(3-33).
Cloning, nucleotide sequence, and expression in Escherichia coli of the exotoxin A structural gene of Pseudomonas aeruginosa.
Authors: Gray G L , Smith D H , Baldridge J S , Harkins R N , Vasil M L , Chen E Y , Heyneker H L
Published: May 2006

A 2760-base pair DNA segment of the Pseudomonas aeruginosa strain PA103 chromosome encoding the exotoxin A (ETA) structural gene has been cloned in Escherichia coli and the nucleotide sequence has been determined. Analysis of the 5'- and 3'-flanking regions indicate that ETA is translated from a monocistronic message. Comparison of the deduced NH2-terminal amino acid sequence with that determined by sequence analysis of the secreted protein indicates that ETA is made as a 638 amino acid precursor from which a highly hydrophobic leader peptide of 25 amino acids is removed during the secretion process. Data are presented that indicate a COOH-terminal location of the ADP-ribosylation activity of the molecule. Expression of the ETA coding sequence in E. coli under control of the E. coli trp promoter, but not the ETA promoter, results in the production of enzymatically and immunologically active protein. However, most of this material appears to be neither processed nor secreted. Comparison of the ETA amino acid and nucleotide sequences to those of diphtheria toxin revealed no significant homologies, indicating that these functionally similar toxins evolved from different genes.
Suppression of phagocytic function and phospholipid metabolism in macrophages by phosphatidylinositol liposomes.
Authors: Wassef N M , Roerdink F , Richardson E C , Alving C R
Published: May 2006

Increased phagocytosis of complement-opsonized vesicles was accompanied by increased phosphatidylinositol (PtdIns) turnover in murine macrophages. However when PtdIns was also present as one of the lipids in the opsonized liposomes, it reduced both phagocytosis and stimulation of endogenous PtdIns turnover. These suppressive effects did not occur with liposomes containing PtdIns phosphate (PtdIns-P). When a monoclonal IgM "anti-PtdIns-P" antibody that bound to inositol phosphate was substituted for antigalactosyl ceramide antibodies for activating complement in the opsonizing process, enhanced phagocytosis occurred normally but increased cellular PtdIns turnover did not occur. Therefore the data show that, although PtdIns-P cannot replace PtdIns for suppressing PtdIns turnover, PtdIns-P can be induced to be suppressive after specific binding to an antibody that recognizes inositol phosphate. We conclude that ingestion of complement-opsonized liposomes by macrophages and complement-induced turnover of cellular PtdIns are separate but related phenomena that can be independently modulated by the polar group of liposomal PtdIns.
Efficient coupled transcription and mRNA splicing in vitro using plasmids derived from early region 3 of adenovirus 2 and a nondefective adenovirus-simian virus 40 hybrid.
Authors: Pawar S , Ahmed C M , Watkins R , Zain S
Published: May 2006

Accurate and highly efficient (80%) splicing of a single mRNA precursor to processed products was achieved using HeLa cell extracts to synthesize and process RNA in vitro from recombinant plasmids containing specific DNA segments from adenovirus 2 (Ad2) and the nondefective adenovirus-simian virus 40 (Ad+2ND1) hybrid. One plasmid, pRID, contains a segment of Ad2 DNA spanning chromosome map coordinates 75.9-83.4. The other plasmid, pRW9, contains the analogous viral region from Ad+2ND1. RNA synthesis from pRID in vitro occurs for more than 60 min and is directed by RNA polymerase II. RNA products consistent in size with the expected precursor and the two processed mRNAs are made. RNA blot hybridization analyses showed that these products are complementary to the Ad2 insert in the plasmid and that the appropriate intervening sequence was absent from the smallest processed mRNA. Comparison of the splice patterns of RNA made in vitro to those of RNAs taken from infected cells using the nuclease S1 technique demonstrated the accuracy of intron removal.
A genetic switch in vitro: DNA inversion by Gin protein of phage Mu.
Authors: Plasterk R H , Kanaar R , van de Putte P
Published: May 2006

Inversion of the G segment in the DNA of Escherichia coli phage Mu depends on the Mu Gin protein and alters the host range of the phage. The frequency of the inversion reaction is low both in the lysogenic state and during lytic growth. A sensitive assay was developed to detect low levels of G inversion: the E. coli lac operon was inserted within the invertible G segment in such a way that the lac operon was expressed only by G(-) clones. As a result Gin-catalyzed inversion from G(+) to G(-) can be monitored as a lactose-negative to lactose-utilizing switch. Using a crude extract from a Gin-overproducing strain and this assay plasmid, we could detect a low level of G inversion in vitro (1% in 30 min). The reaction depends on Mg2+ and a supercoiled substrate. Under optimized reaction conditions over 15% of the plasmids had the G segment inverted after incubation with Gin in vitro. The inversion was then visualized by agarose gel analysis of plasmid DNA digested by restriction endonucleases. The Gin protein retains its catalytic properties upon partial purification. The mechanism of this genetic switch can now be studied in vitro.
Defining erythrocyte internal labeling by phosphorylation.
Authors: Babitch J A , Macha M R , Kiener P A
Published: May 2006

When erythrocytes are incubated with 32Pi, incorporation of label into phosphoproteins is a gradual process, increasing for at least 2 hours. Membrane phospholipids also are labeled. Exogenous protein kinase substrates are unlabeled in these incubations. This suggests that labeling by 32Pi occurs into polypeptides inside the erythrocytes. When erythrocytes are incubated with [gamma-32P]ATP and active protein kinase, membrane polypeptides are not labeled. Only exogenously added protein kinase substrates and the regulatory subunit of protein kinase (and its contaminants) are labeled. This suggests that labeling from [gamma-32P]ATP and active protein kinase occurs in the compartment outside the erythrocytes. Apyrase (EC 3.6.1.5) eliminates such labeling, demonstrating that it was occurring in the compartment external to the erythrocytes. However, in incubations of cells with 32Pi, apyrase has no effect on the incorporation into membrane polypeptides and phospholipids, demonstrating that this labeling occurs on the inside of the membrane. Thus, additions of apyrase to intact particles incubated with protein kinase substrates and 32Pi provides a method for identifying internally exposed polypeptides in the plasma membranes of a variety of systems.
Dimeric structure of single chloride channels from Torpedo electroplax.
Authors: Miller C , White M M
Published: May 2006

The inhibition by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) of Cl- channels from Torpedo electroplax incorporated in planar phospholipid bilayer membranes is studied. DIDS irreversibly and rapidly inhibits the macroscopic conductance of membranes containing many channels. At the single-channel level, the effect of DIDS is more complicated. The uninhibited single channel displays three "substates" of conductances 20, 10, and 0 pS. Short exposure (5-30 s) to 10 microM DIDS converts this three-level active channel into a "conventional" channel of 10-pS conductance. Longer exposure eliminates all channel fluctuations. The results are taken as strong evidence that the Cl- channel is constructed as a functional dimer of identical protein subunits.
Development of methotrexate resistance in a human squamous cell carcinoma of the head and neck in culture.
Authors: Frei E , Rosowsky A , Wright J E , Cucchi C A , Lippke J A , Ervin T J , Jolivet J , Haseltine W A
Published: May 2006

Four methotrexate (MTX)-resistant sublines of a human squamous cell carcinoma (SCC15) were established in culture by progressive dose escalation. The biochemical basis of resistance was studied. The line with the lowest resistance (R1) had a normal dihydrofolate reductase (DHFR) content but showed decreased MTX transport and polyglutamation. Lines of intermediate resistance (R2 and R3) showed an increased DHFR content and DHFR gene copy number and a defect in MTX transport. The line with the greatest resistance (R4) showed increased DHFR content and gene copy number but nearly normal MTX transport. These results demonstrate that multiple mechanisms of MTX resistance occur in human epithelial cells in culture. We also find evidence of alterations in DHFR gene expression. The MTX-resistant cells were either not cross-resistant or only partly cross-resistant to two lipophilic MTX ester derivatives. These compounds are of potential therapeutic interest for the treatment of MTX-resistant tumors.
Use of a replica technique to isolate muscle cell lines defective in expressing the acetylcholine receptor.
Authors: Black R A , Hall Z W
Published: May 2006

We have isolated genetic variants of the C2 muscle cell line that are defective in expressing the acetylcholine receptor (AcChoR). Because the AcChoR is expressed only after C2 myoblasts have fused to form myotubes, we employed a replica technique to detect the variants. This technique yields two copies of each clone, one of which can be used for screening and the other, as a source of dividing cells. In a screening of about 10,000 clones derived from mutagenized cells, we found 2 that fused normally and expressed normal levels of acetylcholinesterase but had reduced amounts of AcChoR on their surface. One of these also had a reduced level of intracellular AcChoR, but, in the other, the amount of intracellular AcChoR was 5-fold higher than normal. Several variants were found that failed to fuse and had reduced levels of both AcChoR and acetylcholinesterase. Though we relied on 125I-labeled alpha-bungarotoxin to distinguish wild-type from deficient clones, we found that an antiserum to the AcChoR, followed by a biotinylated second antibody and a horseradish peroxidase-avidin complex, could also be used. Therefore, it should be possible to obtain muscle cell variants defective in the expression of a variety of proteins for which specific antibodies are available.
Apolipoprotein E mRNA is abundant in the brain and adrenals, as well as in the liver, and is present in other peripheral tissues of rats and marmosets.
Authors: Elshourbagy N A , Liao W S , Mahley R W , Taylor J M
Published: May 2006

The relative amount of apolipoprotein (apo-) E mRNA in 12 different tissues of the rat and marmoset was examined by dot blot hybridization using cloned cDNA probes. As expected, it was found to be most abundant in the liver. However, substantial amounts of apo-E mRNA were found in the brain and adrenals at relative levels about one-third of that found in the liver. Significant quantities of apo-E mRNA were detected in all of the other peripheral tissues as well. The apo-E mRNA levels in these tissues were 2-10% of that found in the liver of the rat and 10-30% of that found in the liver of the marmoset. Apo-E mRNA was also abundant in human brain and in each species examined; it was distributed throughout all major areas of this organ. In contrast, apo-A-I mRNA was detected in abundant amounts only in the small intestine and in the liver. Extrahepatic apo-E mRNA appears to be functional, generating a translation product similar or identical to that generated by the liver. During fetal and neonatal development, apo-E mRNA is rapidly induced from low levels to approximately equal to 60% of adult levels in liver at parturition. The fetal yolk sac contains more apo-E mRNA than the fetal liver, suggesting a significant role for the yolk sac as a source of apo-E during gestation.
Complete nucleotide sequence of an infectious clone of human T-cell leukemia virus type II: an open reading frame for the protease gene.
Authors: Shimotohno K , Takahashi Y , Shimizu N , Gojobori T , Golde D W , Chen I S , Miwa M , Sugimura T
Published: May 2006

The entire nucleotide sequence of an infectious clone of human T-cell leukemia virus type II provirus was determined. This provirus consists of 8952 nucleotides. In addition to long terminal repeats and gag, pol, env, and X, a protease gene that is responsible for processing the gag precursor protein was found. The protease gene is encoded in a different frame from gag and pol and was located between the gag and pol open reading frames. The 5' region of the protease gene overlaps the 3' gag region. Coding regions of the provirus show about 60% homology with those of human T-cell leukemia virus type I at the nucleotide level. The evolutionary relationship between human T-cell leukemia virus types I and II is discussed.
A dimer of AraC protein contacts three adjacent major groove regions of the araI DNA site.
Authors: Hendrickson W , Schleif R
Published: May 2006

Contact sites of AraC protein to the regulatory site araI of the Escherichia coli araBAD operon have been determined by the chemical-interference technique. DNA fragments were chemically modified an average of once per molecule, and fragments that no longer bound AraC were separated by gel electrophoresis from the DNA fragments still able to bind the protein. The contact sites were then determined by comparing the positions of modifications in the two DNA samples. Strong contacts were found with guanines in three consecutive major groove regions and the adjacent phosphates along one side of the DNA. The conserved bases of the AraC-binding DNA consensus sequence are also found in the same positions. The gel electrophoresis assay was used to determine the stoichiometry of binding, and AraC protein was found to bind the araI and araO1 regulatory sites as a dimer. Therefore, AraC appears to bind DNA differently from the other well-characterized regulatory proteins such as phage lambda repressor.
A cII-dependent promoter is located within the Q gene of bacteriophage lambda.
Authors: Hoopes B C , McClure W R
Published: May 2006

We have found a cII-dependent promoter, PaQ, within the Q gene of bacteriophage lambda. Transcription experiments and abortive initiation assays performed in vitro showed that the promoter strength and the cII affinity of PaQ were comparable to the other cII-dependent lambda promoters, PE and PI. The location and leftward direction of PaQ suggests a possible role in the delay of lambda late-gene expression by cII protein, a phenomenon that has been called cII-dependent inhibition. We have constructed a promoter down mutation, paq-1, by changing a single base pair in the putative cII binding site of the promoter by oligonucleotide site-directed mutagenesis. The paq-1 mutant promoter required about 4-fold higher cII concentrations for maximal activation compared to the wild-type PaQ. We tested the hypothesis that PaQ is responsible in part for the delay of lambda late-gene expression by recombining the paq-1 mutation into a phage showing severe cII-dependent inhibition. We found that the paq-1 mutation relieved the cII-dependent growth defect of this phage. The paq-1 mutation (in combination with lambda cI857) resulted in a clear-plaque phenotype at the permissive temperature of 32 degrees C. The role of the PaQ-initiated antisense transcript in the control of lambda development is discussed.
Cloning and sequence analysis of cDNA for the luminescent protein aequorin.
Authors: Inouye S , Noguchi M , Sakaki Y , Takagi Y , Miyata T , Iwanaga S , Miyata T , Tsuji F I
Published: May 2006

The luminescent jellyfish Aequorea contains a photoprotein, aequorin, which emits light by an intramolecular reaction in the presence of a trace amount of Ca2+. A cDNA library of Aequorea was constructed and clones carrying the cDNA for the Ca2+-dependent photoprotein were isolated by the method of colony hybridization using synthetic oligonucleotide probes. The primary structure of the protein deduced from the nucleotide sequence showed that the protein is composed of 189 amino acid residues and has three E-F hand structures that are characteristic for Ca2+-binding sites. The sequence also suggested that the protein has hydrophobic regions at which the protein may interact with its functional chromophore, coelenterazine.
Animal cell mutants defective in glycosaminoglycan biosynthesis.
Authors: Esko J D , Stewart T E , Taylor W H
Published: May 2006

We have obtained Chinese hamster ovary cell mutants defective in the biosynthesis of glycosaminoglycans by screening replicate colonies immobilized on polyester cloth. Depending upon the strain, the mutants accumulated less 35S-labeled glycosaminoglycans per microgram of cell protein by a factor of 6-60 compared to the wild type. Some of the mutants incorporated [6-3H]glucosamine into glycosaminoglycans to the same extent as the wild type, suggesting that sulfate addition was specifically altered. In contrast, five strains failed to generate 3H-labeled glycosaminoglycans normally. In four of these, the initiation of glycosaminoglycan assembly was specifically altered, since the addition of p-nitrophenyl-beta-xyloside restored sulfation to normal. Enzymatic assay of the xylosyltransferase in extracts prepared from these mutants revealed that one of the strains, S745, contained less enzyme activity by a factor of 15 than the wild type. This mutant provides genetic evidence that the xylosyltransferase assayed in vitro is responsible for the initiation of chondroitin sulfate and heparan sulfate biosynthesis in vivo.
Differential expression of mitochondrial genes between life cycle stages of Trypanosoma brucei.
Authors: Feagin J E , Stuart K
Published: May 2006

The mitochondrial respiratory system is differentially produced during the life cycle of the parasitic protozoan, Trypanosoma brucei. We have found 14 transcripts that are derived from a maxicircle (mitochondrial DNA) region that contains sequences homologous to cytochrome c oxidase subunits I and II, unassigned reading frame 1 from mitochondrial DNA of other organisms, and two other open reading frames. Ten of these transcripts occur as pairs that differ in size by approximately equal to 200 nucleotides. While most of these transcripts occur in both life cycle forms, four transcripts are differentially expressed. Of these, two are more abundant in bloodstream forms, one is more abundant in procyclic forms, and one is present in procyclic but not bloodstream forms. These results indicate that the differential production of the mitochondrial respiratory system in T. brucei entails regulation of mitochondrial transcriptional and/or posttranscriptional processes.
Thrombus radioimmunoscintigraphy: an approach using monoclonal antiplatelet antibody.
Authors: Oster Z H , Srivastava S C , Som P , Meinken G E , Scudder L E , Yamamoto K , Atkins H L , Brill A B , Coller B S
Published: May 2006

Thrombus detection and localization is of cardinal importance in clinical medicine. The currently available method using autologous 111In-labeled platelets is too lengthy and complex for everyday use. It requires careful separation of the platelets prior to labeling and visualization of the thrombus becomes possible only 24 hr after injection. An approach to thrombus imaging using a monoclonal antiplatelet antibody labeled with 111In or 123I is described. The antibody (7E3) prepared against human platelets inhibits the interaction between fibrinogen-coated beads and both human and dog platelets. 7E3 is an IgG1 that binds to the complexed glycoprotein IIb/IIIa. Ninety percent of a tracer dose of radiolabeled 7E3 binds to human platelets and 50% binds to dog platelets. In vitro studies showed that virtually all of the platelet-bound radioactivity becomes incorporated into clots formed by adding thrombin to whole blood. 7E3 was labeled with 111In by the cyclic anhydride diethylenetriaminepentaacetic acid method or by radioiodination with 123I. At a ratio of 1:50 (anhydride:7E3) the specific activity ranged between 10 and 40 muCi/micrograms (1 Ci = 37 GBq) without change in the antibody characteristics. In vivo studies in dogs were performed by preincubating for 1 hr the radiolabeled 7E3 with citrated blood or by directly injecting the radiolabeled 7E3 intravenously. Experimental thrombi were induced by transcatheter placement of copper coils into peripheral arteries and veins as well as in the superior vena cava and pulmonary artery. With gamma camera, visualization of venous and arterial thrombi as well as sites of intimal injury without visible thrombi, could be observed 1-1.5 hr after injection. There was no need for delayed imaging because of the fast clearance of radioactivity from the circulation nor was there need for blood pool subtraction. Two to 10-hr thrombi could be imaged but 48-hr thrombi were not detectable with this method. No change in platelet counts before and after the injection of labeled 7E3 nor increased bleeding tendency occurred. The advantages of this method are a shorter preimaging preparation time, faster visualization after injection, and no need for delayed imaging or subtraction techniques. For these reasons human investigations seem to be warranted.
Immunological properties of the Gag protein p24 of the acquired immunodeficiency syndrome retrovirus (human T-cell leukemia virus type III).
Authors: Sarngadharan M G , Bruch L , Popovic M , Gallo R C
Published: May 2006

Antigenic cross-reactivity of human T-cell leukemia virus type III (HTLV-III) with HTLV-I and HTLV-II and other retroviruses was measured by using a stringent homologous competition radioimmunoassay for the Gag protein p24 and a less stringent electrophoretic transfer blot assay. In the competition radioimmunoassay only minimal cross-reactivities were detected between HTLV-III p24 and both HTLV-I and HTLV-II. No cross-reactivity was detected with any other retrovirus. In the electrophoretic transfer blot system using rabbit antibody to HTLV-I, HTLV-II, and HTLV-III, low-level cross-reaction was detected between HTLV-I and HTLV-III and between HTLV-II and HTLV-III. Unlike the cross-reactivity between HTLV-I p24 and HTLV-III p24, which was bidirectional, the one between HTLV-II and HTLV-III was only a one-way reactivity. Antiserum to HTLV-II recognized HTLV-III p24, but the antiserum to HTLV-III did not recognize HTLV-II p24. The results indicate that HTLV-III is a unique retrovirus with a limited homology with HTLV-I and HTLV-II but unrelated to most other retroviruses.