bluuebunny/crossref_metadata_2025 huggingface dataset:
Organization of a hybrid between phage f1 and plasmid pSC101.
Authors: Ravetch J V , Ohsumi M , Model P , Vovis G F , Fischhoff D , Zinder N D
Published: May 2006

We have characterized the 200-nucleotide-long insertion found in f1 after segregation of a chimeric phage containing the genomes of f1 and pSC101 [Ohsumi, M., Vovis, G.F. & Zinder, N.D. (1978) Virology 89, 438--449]. The insertion in this novel f1 species, called f1', is derived from pSC101 and has the potential to form an extended base-paired secondary structure, as determined by nucleotide sequence analysis. A five-nucleotide direct repeat, derived from f1 sequences, is present in f1'. The 200 additional nucleotides that are inserted into the DNA sequence coding for the carboxy terminus of f1 gene IV protein have generated a novel carboxy terminus for the f1' gene IV protein. In vitro transcription--translation studies demonstrate that a read-through protein can be expressed, as predicted from the f1' nucleotide sequence results. This 200-nucleotide-long sequence appears to be a transposable element found within pSC101 and is similar in sequence to the inverted repeat found in Tn3. Restriction enzyme analysis of the chimeric phage DNA, coupled with the nucleotide sequencing results, allows us to predict a structure for the genomic organization of this chimera.
Distinct single-copy sequence sets in sea urchin nuclear RNAs.
Authors: Ernst S G , Britten R J , Davidson E H
Published: May 2006

The purpose of this study was to determine whether nuclear RNAs (nRNA) of sea urchin embryos and adult tissues contain identical or partially distinct sets of single-copy sequence transcripts. A DNA tracer was prepared consisting mainly of sequences absent from gastrula nRNA; 3.6% of this tracer reacted with adult intestine nRNA but not with gastrula nRNA. The existence of a differentially transcribed DNA fraction was verified by its partial purification and rehybridization to intestine and gastrula nRNAs. About one-third of the genomic single-copy sequence is represented in both nRNAs, or about 2 x 10(8) nucleotides. The differentially transcribed portion of the single-copy genome identified in this work includes about 3.5 x 10(7) nucleotides.
Synthesis and use of 8-azidoguanosine 3',5'-cyclic monophosphate as photoaffinity label for cyclic GMP-dependent protein kinase.
Authors: Geahlen R L , Haley B E , Krebs E G
Published: May 2006

8-Azidoguanosine 3',5'-cyclic monophosphate (8-N3cGMP) has been synthesized for use as a photoactive probe for the labeling of cGMP receptors. The ability of 8-N3cGMP to be bound at specific cGMP binding sites was demonstrated by its ability to activate cGMP-dependent protein kinase isolated from bovine lung (Ka = 1.1 x 10(-7) M) and to inhibit competitively the binding of [3H] cGMP to the enzyme [Kd (8 N3GMP)/Kd (cGMP) = 6]. Photolysis of 8-N3[32P]cGMP in the presence of a crude enzyme preparation resulted in the covalent attachment of analog to cGMP-dependent protein kinase. Half-maximal labeling occurred at 2.2 x 10(-7) M. The incorporation of the analog was completely inhibited by the addition of cGMP.
Transcription program of the chloroplast genome of
Euglena gracilis
during chloroplast development
Authors: Chelm Barry K. , Hallick Richard B. , Gray Patrick W.
Published: May 2006

RNA transcription in
Euglena gracilis
chloroplasts has been characterized by hybridization of RNA from cells at different stages of chloroplast development to
3
H-labeled chloroplast DNA restriction endonuclease fragments. Chloroplast DNA was digested into five fragments of 53,000, 35,000, 25,000, 10,000, and 6900 base pairs with
Pst
I. The 53,000-base-pair DNA was further cleaved by
Bam
HI digestion. Eight different DNA fragments, of known restriction nuclease map location and accounting for the entire genome, were labeled
in vitro
by means of the nick translation reaction of DNA polymerase I with [
3
H]dTTP as a substrate. RNA was isolated from dark-adapted
Euglena
cells, and from cells at an early (4 hr) and a late (72 hr) stage of light-induced chloroplast development. The RNAs were hybridized in solution to each
3
H-labeled chloroplast DNA fragment. From the extents and kinetics of the reactions, a temporal program for RNA transcription from defined regions of the chloroplast genome could be described. Different classes of transcription units are present, including RNAs (
i
) continuously present throughout development, (
ii
) induced at the onset of development, (
iii
) repressed early in development, and (
iv
) induced late in development.
Preferential digestion of basement membrane collagen by an enzyme derived from a metastatic murine tumor.
Authors: Liotta L A , Abe S , Robey P G , Martin G R
Published: May 2006

The specificity of human skin collagenase and of an enzyme from an invasive tumor were studied by using types I, II, III, IV, and V (AB) collagen as substrates. Human skin collagenase degraded types I, II, and III collagen, producing the characteristic 3/4 and 1/4 cleavage products, but failed to degrade type IV or V collagen. Collagenase prepared from the invasive tumors showed maximal activity after trypsin treatment. The tumor enzyme degraded type IV (basement membrane) collagen, producing fragments consistent with a single cleavage site but did not attack types I, II, III, and V collagen. Because type IV collagen prepared by pepsinization of placenta was also digested, it is likely that cleavage of type IV collagen by the tumor collagenase occurs within a largely helical domain. A type IV collagenase could play a significant role in tumor metastases and in normal tissues where basement membrane turnover takes place.
A genetic approach to analysis of transposons.
Authors: Beck C F
Published: May 2006

Integration of the tetracycline resistance transposon Tn10 into lacI of a lacI-lacZ gene fusion permits the isolation of deletions that excise DNA from one end of Tn10 and fuse Tn10 genes with lacZ in such a manner that chimeric proteins with beta-galactosidase activity are produced. The synthesis of the chimeric proteins is under the same control as the transposon genes. Thus, regulation of expression of Tn10 genes can be investigated by measuring beta-galactosidase activity. Analysis of Tn10-lacZ fusions revealed different deletion endpoints within Tn10; lacZ has been fused to at least three different Tn10 genes or operons. Two of these genes are under the control of a tetracycline repressor.
Septum formation, cell division, and sporulation in mutants of yeast deficient in proteinase B.
Authors: Zubenko G S , Mitchell A P , Jones E W
Published: May 2006

Mutants of Saccharomyces cerevisiae that carry lesions in the structural gene for proteinase B, prb1, have been isolated. Proteinase B was thought to be involved in activation of chitin synthase zymogen to allow septum formation during budding and to be involved in sporulation. The prb1 mutants are able to grow and divide and they form apparently normal septa that contain chitin. Most diploids homozygous for prb1 mutations are unable to complete sporulation.
Pregnant mice are not primed but can be primed to fetal alloantigens.
Authors: Wegmann T G , Waters C A , Drell D W , Carlson G A
Published: May 2006

In this report we present evidence gathered from various stages of murine pregnancy that indicates that pregnant females have no demonstrable immune effector function directed against their semiallogeneic fetuses. Specifically, by using in vitro assays we found that cytotoxic lymphocytes were not present in pregnant mice, and pregnant mice challenged with radiolabeled paternal strain leukemia cells showed no evidence of immune elimination. Nonetheless, immunity measurable both in vitro and in vivo was readily induced by priming with the paternal strain cells. No harm to the fetus was observed in primed mothers. These results cast doubt on the relevance of mechanisms proposed that involve systemic active suppression during pregnancy. The results are compatible with the hypothesis that the placenta acts as a barrier, preventing fetal cells from priming the mother.
Simultaneous hermaphroditism and sexual selection
Authors: Charnov Eric L.
Published: May 2006

Theory about the evolution of sexual behavior in dioecious species is based on the general assumption that egg production is limited by a female's ability to garner resources to make eggs, not by a lack of sperm to fertilize them. Reproductive success for males is thus limited by access to females (and their eggs). I suggest that egg production by simultaneous hermaphrodites also obeys this principle—that fertilized egg production by an individual is not limited by sperm availability, but by resources allocated to eggs. If true, this suggests that sperm competition (reproduction success through male function) and a form of male—female conflict have played important roles in the evolution of hermaphroditism.
Determination of enzyme mechanisms by radiationless energy transfer kinetics
Authors: Lobb Roy R. , Auld David S.
Published: May 2006

Rigorous definition of the elementary steps of an enzymatic reaction requires visualization of transient enzyme—substrate (ES) complexes. Measurement of radiationless energy transfer (RET) between enzyme tryptophan residues and a fluorescent dansyl (5-dimethylaminonaphthalene-1-sulfonyl) substrate provides a sensitive means to observe ES complexes directly. Analysis of the rate of formation and breakdown of ES complexes by RET can serve as the basis of a rapid kinetic approach to enzyme mechanisms. Both pre-steady-state and steady-state kinetics can be performed in the same RET experiment. Analysis at steady state precisely determines
k
cat
and
K
m
values by multiple means. Analysis at pre-steady state determines the number of intermediates, the type of reaction mechanism, and all the individual binding and rate constants. Chymotrypsin was chosen as a standard of reference for RET kinetics because extensive investigations have established both the existence of transient intermediates in the course of its catalytic process and the range of values to be expected for pertinent kinetic constants. As predicted, RET kinetics readily detects the two known intermediates in the α-chymotrypsincatalyzed hydrolysis of specific ester substrates. The results are both qualitatively and quantitatively in accord with data derived for this enzyme from classical kinetics. Hence, this experimental study both validates and demonstrates the theoretical advantages and potential of RET kinetics. The generality of the approach has been investigated by synthesizing a family of dansyl-labeled substrates designed to meet the specificity requirements of a number of metallo- and nonmetallo- exo- and endopeptidases. In all cases, the ES complex is observed readily at micromolar or lower concentrations of enzyme under stopped-flow conditions. The success of the RET kinetic approach on proteolytic enzymes shows its broad utility.
De novo biosynthesis of an enzymatically active precursor form of bovine pancreatic RNase.
Authors: Haugen T H , Heath E C
Published: May 2006

The de novo biosynthesis of RNase (ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) was studied in a cell-free rabbit reticulocyte translation system using a polyadenylylated fraction of mRNA isolated from bovine pancreas. Analysis of the [35S]methionine-labeled translation products of pancreas mRNA by polyacrylamide gel electrophoresis indicated the presence of several proteins, one of which corresponded to 16,500 daltons, or approximately 2800 daltons greater than native RNase A. This protein was specifically precipitated from the mixture of products by anti-RNase antibodies. Partial sequence determination of the NH2-terminal region of the anti-RNase antibody-precipitable species indicated that it is a precursor form of RNase A with 25 additional amino acids on its NH2 terminus. The precursor nature of the protein was confirmed by demonstration that a mixture of RNase A and a glycosylated form of the enzyme, RNase B, is formed when translation of the mRNA is conducted in the presence of dog pancreas membranes. Assay of the putative precursor form of RNase for catalytic activity with polycytidylic acid as substrate indicated that the protein has a specific enzymatic activity identical to that of native RNase A.
Human promyelocytic leukemia cells in culture differentiate into macrophage-like cells when treated with a phorbol diester.
Authors: Rovera G , Santoli D , Damsky C
Published: May 2006

When suspension cultures of human promyelocytic leukemia cells (line HL60) were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA; 1.6-160 nM), more than 80% of the cells adhered to the plastic substrate within 24 hr. Within the same time period the immature azurophilic granulations typical of HL60 promyelocytic cells disappeared and the nuclear chromatin became more condensed, but the nucleolus was retained. The attached cells stopped dividing and synthesizing DNA. The phenomenon was irreversible and independent of the continuous presence of TPA. Approximately 60% of the untreated cells and of TPA-treated cells bore surface Fc receptors for IgG. Under the experimental conditions used, about 10% of the TPA-treated cells were also able to phagocytize IgG-coated erythrocytes and more than 80% were able to phagocytize latex beads, but untreated controls were unable to do so. Cellular levels of NADase, acid phosphatase, and non-specific esterase were markedly increased after treatment with TPA, whereas little or no increase was seen after treatment with dimethyl sulfoxide (Me2SO), a drug that induces myeloid differentiation of HL60 cells. Peroxidase activity was lower in TPA-treated and Me2SO-treated cells than in HL60 cells. More lysozyme was found in the medium of TPA-treated cells than in the medium of untreated or Me2SO-treated cells. These data indicate that, after treatment with TPA, human promyelocytic leukemia cells can differentiate into cells that have several characteristics of macrophages.
AU cell-surface antigen of human malignant melanoma: solubilization and partial characterization.
Authors: Carey T E , Lloyd K O , Takahashi T , Travassos L R , Old L J
Published: May 2006

AU antigen is defined by reactions of sera from patient AU with cell-surface antigens of cultured autologous melanoma cells (SK-MEL-28). Past studies established that no available cell type other than AU melanoma expressed AU antigen. By use of antibody inhibition tests for antigen detection, limited papain digestion of AU melanoma cells was found to result in the solubilization of AU antigen along with beta2-microglobulin (beta 2m) and HLA allogeneic and xenogeneic specificities. Comparable papain treatment of other melanoma and non-melanoma cell lines solubilized beta 2m and HLA, but did not result in the release of antigen with AU reactivity. Maximum yield of AU antigen from AU melanoma cells was obtained after very short (5-15 min) digestion times in contrast to the more prolonged proteolysis required for maximum HLA and beta 2m release. AU antigen was not immunoprecipitated by rabbit antiserum against beta 2m or HLA under conditions leading to partial or complete removal of beta 2m and HLA. At least a proportion of the molecules with AU determinants appear to be glycoproteins, as indicated by specific affinity for Lens culinaris hemagglutinin (LcH). After affinity chromatography on LcH-agarose, the specific activity of AU antigen was increased 50-fold. As determined by gel filtration chromatography, AU antigen has a molecular weight in the range of 20,000-50,000.
Partial purification of Rho (D) antigen from Rh positive and negative erythrocytes.
Authors: Plapp F V , Kowalski M M , Tilzer L , Brown P J , Evans B J , Chiga M
Published: May 2006

A rapid method is described for partial purification of Rho(D) antigen from sodium deoxycholate-solubilized erythrocyte membranes by affinity chromatography on a column coupled with anti-Rho(D) IgG. The Rho(D) antigen is a low molecular weight membrane protein that comigrates with the lipid zone of erythrocytes during sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Similar quantities of Rho(D) antigen were present in Rh positive and negative erythrocytes. However, in the latter erythrocytes the antigen may not be exposed to the external cell surface, explaining why these cells are not agglutinated by anti-Rho(D) antiserum. LW antigen was also present in the fraction containing Rho(D) antigen. The genetic implications of these findings are discussed.
Determination of water mass properties of water pumped down from the Ekman layer to the geostrophic flow below
Authors: Stommel Henry
Published: May 2006

In a subtropical gyre, the convergent Ekman layer forces water downward into the geostrophic flow below. The properties and depth of the mixed layer vary considerably during the course of the year, but this variability does not penetrate into the geostrophic region. Evidently there is some process at work that selects only late winter water for actual net downward pumping. It is a process much like that performed by Maxwell's Demon. Arguing from a particular example in a North Atlantic region where Sverdrup dynamics is presumed to prevail, an elementary description is given as to how the Demon works and a sample set of profiles by season is computed. Present day mixed layer models involve a local vertical mixing process. Thermocline theories involve adiabatic flow from high latitudes. There is an intermediate depth range at which the two processes operate alternatively, according to season.
Active transport of calcium in Neurospora plasma membrane vesicles.
Authors: Stroobant P , Scarborough G A
Published: May 2006

Functionally inverted plasma membrane vesicles isolated from the eukaryotic microorganism Neurospora crassa catalyze Mg2+/ATP-dependent Ca2+ uptake. Inhibitors induced efflux studies and isotope-exchange experiments indicate that the Ca2+ is accumulated inside the vesicles against a concentration gradient of about 40-fold, and that the majority of the transported Ca2+ is present essentially in free solution. Comparisons of Mg2+/ATP-driven 45Ca2+ uptake and [14C]SCN-uptake with respect to the Mg2+/ATP concentration dependence, the effects of inhibitors, and the nucleotide and divalent cation specificities indicate that the energy for Ca2+ accumulation is derived from ATP hydrolysis catalyzed by the electrogenic plasma membrane ATPase. Energized Ca2+ uptake is stimulated by the permeant anion SCN- to a degree that varies reciprocally with the ability of this anion to dissipate the membrane potential, and is inhibited by K+ in the presence of nigericin. All of these data point to the conclusion that the active transport of Ca2+ across the Neurospora plasma membrane takes place via a Ca2+/H+ antiporter, which functions to pump Ca2+ out of the intact cell.
Sites of intermolecular crosslinking of fatty acyl chains in phospholipids carrying a photoactivable carbene precursor
Authors: Gupta Chhitar M. , Costello Catherine E. , Khorana H. Gobind
Published: May 2006

Sonicated vesicles of 1-fatty acyl-2-ω-(2-diazo-3,3,3-trifluoropropionoxy) fatty acyl
sn
-glycero-3-phosphoryl-cholines were shown recently to form intermolecular crosslinks by insertion of the photogenerated carbene into a C—H bond of a neighboring hydrocarbon chain. We now report that photolysis of multilamellar dispersions gives a second series of products in which carbene insertion is accompanied by elimination of a molecule of hydrogen fluoride. The sites of crosslinking in the latter compounds have been studied by mass spectrometry using phospholipids with varying chain lengths of the fatty acyl groups carrying the carbene precursor. The patterns observed show that the point of maximum crosslinking is consistent with the recent conclusion that in phospholipids the
sn
-2 fatty acyl chain trails the
sn
-1 chain by 2-4 atoms.
Glucagon-induced autophagy and proteolysis in rat liver: mediation by selective deprivation of intracellular amino acids.
Authors: Schworer C M , Mortimore G E
Published: May 2006

Amino acid deprivation and glucagon are both potent inducers of autography and proteolysis in liver. Because glucagon enhanced the metabolic utilization of some amino acids, the catabolic response to both of these stimuli could be achieved by a lowering of intracellular amino acid pools. Alternatively, glucagon could act independently of amino acids. To clarify the mode of hormonal action and also the relationship between the two cellular responses, livers from fed rats were perfused, with and without glucagon, with plasma amino acids over a concentration range of 0 to 10 times normal. Individual amino acids constancy at each level was ensured by perfusion in the single-pass mode. Amino acids alone strongly regulated autophagy and proteolysis in a coordinated fashion; maximal suppression was achieved at twice normal concentration; both effects increased rapidly to maximum at less than normal concentration. Corresponding effects of glucagon, however, could be elicited only at intermediate amino acid levels. None was noted at 4 and 10 times normal; at 0, hormonal stimulation was minimal. The amino acid inhibition was selective because it did not block cyclic AMP production or glycogenolysis. Intracellular pool measurements and systematic alteration of perfusate amino acid composition indicated that the autophagic and proteolytic effects of glucagon are mediated by a hormonally induced depletion of glycine, alanine, glutamate, and glutamine; of these, glutamine alone is the most effective. We conclude that the stimulation of intracellular protein degradation in liver is a manifestation of deprivation-induced autophagy which results from a decrease in certain intracellular glucogenic amino acids, notably glutamine.
Uptake and accumulation of putrescine and its lethality in Anacystis nidulans.
Authors: Guarino L A , Cohen S S
Published: May 2006

The rate of uptake of putrescine by Anacystis nidulans has been shown to depend on the external pH and the extracellular concentration of putrescine. Accumulation of exogenous putrescine was also proportional to the concentration of putrescine in the medium, suggesting that putrescine uptake was not subject to cellular regulation. An equation was derived to test the hypothesis that putrescine accumulation was due to ion trapping. Comparison of the predicted and observed intracellular concentrations of putrescine under various conditions showed a close correlation in support of the hypothesis of ion trapping. Under conditions leading to cell death (e.g., 150 microM putrescine, pH 9.8), the correlation did not hold as a result of leakage of accumulated putrescine.
Entrapment of a bacterial plasmid in phospholipid vesicles: potential for gene transfer.
Authors: Fraley R T , Fornari C S , Kaplan S
Published: May 2006

Entrapment of pBR322 DNA within liposomes was demonstrated by (i) its comigration with liposomes on Sepharose 4B columns, (ii) resistance of its biological activity to DNase digestion, and (iii) identification of plasmid DNA on agarose gels after lipid extraction. The biological activity of the liposome-entrapped plasmid was determined by transformation assays. The incubation of intact liposomes, containing entrapped pBR322, with competent Escherichia coli cells in the standard transformation mixture resulted in the appearance of tetracycline-resistant colonies at a frequency of 1% of the control frequency. Importantly, this frequency was unaffected by the addition of DNase to the incubation mixture, whereas transformation by free pBR322 DNA was totally eliminated after treatment with DNase.
Sustained growth and three-dimensional organization of primary mammary tumor epithelial cells embedded in collagen gels.
Authors: Yang J , Richards J , Bowman P , Guzman R , Enami J , McCormick K , Hamamoto S , Pitelka D , Nandi S
Published: May 2006

We have developed a method for embedding cells within a collagen matrix which allows sustained growth of mouse mammary tumor epithelial cells in primary culture. A characteristic and reproducible pattern of organization and growth occurs: the cells rearrange themselves and produce duct-like structures extending into the matrix, resulting in a three-dimensional outgrowth. Autoradiography showed continuous [3H]thymidine incorporation during 8 weeks in culture. An increase in DNA content of the cultured cells as a function of time was observed. Mouse mammary tumor cells cultured in the conventional monolayer system failed to show any significant increase in cell number during a culture period of 6 weeks. In addition, in such monolayer systems, cells progressively became detached from the dishes in long-term culture. The mammary epithelial cell origin of the collagen gel cell outgrowths was shown by electron microscopic demonstration of polarized cells containing tight junctions and budding mammary tumor virus particles. In addition, in vivo transplantation of collagen gel outgrowths resulted in the development of mammary adenocarcinoma histologically similar to the donor tumor. Cellular outgrowth patterns resembling those from tumor cells were also seen in similar collagen gel cultures of normal mammary cells from mouse and human and of hyperplastic alveolar nodule cells from mouse. The significance and usefulness of this system in comparison to the conventional monolayer system are discussed.
Abnormalities in clonable B lymphocytes and myeloid progenitors in autoimmune NZB mice.
Authors: Kincade P W , Lee G , Fernandes G , Moore M A , Williams N , Good R A
Published: May 2006

Cloning procedures were used to study B lymphocytes and progenitors of granulocytes and macrophages in NZB mice. Numbers of B cells that were detected in sheep erythrocyte-containing semisolid cultures were only slightly elevated in NZB tissues, and these were normally sensitive to inhibition by anti-mu or anti-delta antibodies or prostaglandin E. However, NZB mice rapidly developed large numbers of B cells that could be cloned in the presence of lipopolysaccharide, and these included unusual anti-mu resistant cells. Numbers of myeloid precursors in NZB bone marrow that were responsive to colony-stimulating activity in L-cell conditioned medium or endotoxin serum were at least normal, but at all ages granulocyte-macrophage precursors were poor responders in cultures stimulated by WEHI-3 cell conditioned medium. Almost no colonies were elicited in NZB cultures with a colony-stimulating activity moiety from WEHI-3 cells. Prostaglandin sensitivity of myeloid precursors from NZB and CBA mice was also different. Codominant genetic control of these abnormalities was suggested by their partial expression in F1 hybrid NZB X CBA and NZB X NZW mice. NZB mice expressed an unexpected IgD allotype allele.
Attempt to immunize guinea pigs against L2C leukemia with leukemia cells inactivated by gamma irradiation.
Authors: Gross L , Dreyfuss Y
Published: May 2006

In previous studies, intradermal inoculation of small doses of L2C leukemic cell suspensions into strain 2 guinea pigs induced immunity against challenging reinoculation with leukemic cells; however, 50% of the guinea pigs developed leukemia in the course of immunization. We have now attempted to induce immunity by inoculation of L2C leukemic cells inactivated by in vitro gamma irradiation ranging from 750 to 8000 rads (1 rad = 0.01 gray). In preliminary experiments, irradiation with 750 to 2750 rads had no significant effect on leukemogenic potency of leukemic cells; however, doses exceeding 3000 rads inactivated the leukemogenic potency of L2C cells. Eighty-nine guinea pigs that survived intradermal, subcutaneous, or intraperitoneal inoculations with irradiated (1000-8000 rads) L2C cells were subsequently challenged by reinoculation with nonirradiated leukemic cells, and 83 of them (93%) developed leukemia. L2C leukemic cells contain spherical particles, about 103 nm in diameter; it is reasonable to assume that these particles represent the causative virus responsible for the development of L2C leukemia. Inactivation of leukemogenic potency of L2C leukemic cells by gamma irradiation in vitro does not necessarily imply that the virus particles consistently present in these cells were also inactivated. In previous experiments carried out on mouse leukemia, doses exceeding 1,000,000 rads were needed in order to inactivate the mouse leukemia virus in vitro.
Pyrazoles as inhibitors of alcohol oxidation and as important tools in alcohol research: An approach to therapy against methanol poisoning
Authors: Blomstrand Rolf , Östling-Wintzell Helene , Löf Agneta , McMartin Kenneth , Tolf Bo-Ragnar , Hedström Karl-Göran
Published: May 2006

4-Methylpyrazole, in a dose producing inhibition of alcohol dehydrogenase (alcohol:NAD
+
oxidoreductase, EC 1.1.1.1), was given alone or together with ethanol (10%) as sole drinking fluid to growing rats for up to 38 weeks. Their weight curves remained normal. Electron microscopy of liver, kidney, and heart revealed no changes related to treatment. Hematologic analysis showed normal values for blood and bone marrow. Several clinical chemical parameters showed no impairment of liver or kidney function, except for an enhancement of the microsomal drug-metabolizing activity after concurrent administration of 4-methylpyrazole and ethanol. A study on rats receiving 4-methylpyrazole and ethanol indicated a mutual interaction of the two compounds or the metabolites, leading to increased concentration in the blood of the compounds and reduced formation of 4-hydroxymethylpyrazole, the primary metabolite of 4-methylpyrazole. In monkeys, elimination of 4-methylpyrazole followed a linear course. 4-Hydroxymethylpyrazole accumulated to a level of at most 10% of that of 4-methylpyrazole. Concurrent administration of methanol inhibited the elimination of 4-methylpyrazole about 25%, and 4-methylpyrazole produced a profound inhibition of the oxidation of methanol. 4-Methylpyrazole, at a level in the plasma of more than 10 μM, prevented accumulation of the toxic metabolite formic acid in methanol-poisoned monkeys, and repeated injections of 4-methylpyrazole abolished methanol toxicity in monkeys receiving lethal doses of methanol. The present investigation indicates that 4-methylpyrazole, with its low toxicity and strong inhibition of alcohol oxidation, is a valuable tool for experimental studies of alcohol metabolism and its effects. It illustrates the usefulness of the monkey as a model to study 4-methylpyrazole activity and toxicity in light of its possible use for treating methanol poisoning in human beings.
The gamma-aminobutyric acid system in rabbit retina: localization by immunocytochemistry and autoradiography.
Authors: Brandon C , Lam D M , Wu J Y
Published: May 2006

The localization of gamma-aminobutyric acid (GABA) neurons in the rabbit retina has been studied by immunocytochemical localization of the GABA-synthesizing enzyme L-glutamate decarboxylase (L-glutamate I-carboxy-lyase, EC 4.1.1.15) and by [3H]GABA uptake autoradiography. When Triton X-100 was included in immunocytochemical incubations with a modified protein A-peroxidase-antiperoxidase method, reaction product was found in four broad, evenly spaced laminae within the inner plexiform layer. In the absence of the detergent, these laminae were seen to be composed of small, punctate deposits. When colchicine was injected intravitreally before glutamate decarboxylase staining, cell bodies with the characteristic shape and location of amacrine cells were found to be immunochemically labeled. Intravitreally administered [3H]GABA produced a diffuse labeling of the inner plexiform layer and a dense labeling of certain amacrine cell bodies in the inner nuclear layer. Both immunocytochemical and autoradiographic results support the notion that certain, if not all, amacrine cells use GABA as their neurotransmitter.
Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate.
Authors: Wahl G M , Stern M , Stark G R
Published: May 2006

We describe a technique for transferring electrophoretically separated bands of double-stranded DNA from agarose gels to diazobenzyloxymethyl-paper. Controlled cleavage of the DNA in situ by sequential treatment with dilute acid, which causes partial depurination, and dilute alkali, which causes cleavage and separation of the strands, allows the DNA to leave the gel rapidly and completely, with an efficiency independent of its size. Covalent attachment of DNA to paper prevents losses during subsequent hybridization and washing steps and allows a single paper to be reused many times. Ten percent dextran sulfate, originally found to accelerate DNA hybridization in solution by about 10-fold [J.G. Wetmur (1975) Biopolymers 14, 2517-2524], accelerates the rate of hybridization of randomly cleaved double-stranded DNA probes to immobilized nucleic acids by as much as 100-fold, without increasing the background significantly.
Nicking-closing activity associated with bacteriophage lambda int gene product.
Authors: Kikuchi Y , Nash H A
Published: May 2006

Integrative recombination of bacteriophage lambda requires the action of the protein Int, the product of the phage int gene. In this paper we show that highly purified Int relaxes supercoiled DNA. The association of this nicking-closing activity with Int is shown by: (i) the cosedimentation of nicking-closing and recombination activities of purified Int, (ii) the parallel inactivation of the two activities in purified Int by both heat and a specific antiserum, and (iii) the alteration of both activities in crude extracts of a strain expressing a mutant int gene. The nicking-closing activity of Int functions in the absence of divalent cations and in the absence of an apparent source of chemical energy. The activity displays no obvious sequence specificity and is inhibited by Mg2+, spermidine, and single-stranded DNA. Int relaxes positive as well as negative supercoils. We present a model for the mechanism of strand exchange that describes how the nicking-closing activity of Int might be used during recombination.
Ratio of tree branch lengths: The equitable distribution of leaf clusters on branches
Authors: Honda Hisao , Fisher Jack B.
Published: May 2006

The theoretical optimal ratios of branch lengths, which produce the most equitable distribution (minimum deviation) of leaf clusters in a computer-stimulated branch system, are similar to the observed ratios in real trees of
Terminalia
. In another study, observed values for branch angles in this species were shown to be similar to theoretical optimal values that produce the maximum effective leaf area. The relationship between these two fundamental parameters of bifurcation and tree geometry and their effect on effective leaf area and distribution of leaves are discussed.
Thymus-derived lymphocytes control the expression of immunogenic properties of peritoneal macrophages.
Authors: Tzehoval E , Segal S , Feldman M
Published: May 2006

Macrophages, given keyhole limpet hemocyanin in vitro, from normal mice initiate in culture antigen-specific T cell-mediated immune reactions. On the other hand, macrophages from nude, from adult-thymectomized, or from neonatal-thymectomized mice are impaired with respect to their capacity to signal such an antigen-specific T cell reaction. Thymocytes from hydrocortisone-treated donors, added in culture to such impaired macrophages, rendered them immunologically potent. The capacity of macrophages from adult thymectomized mice to promote the activation of antigen-specific "helper" T cells which, cooperating with B lymphocytes, would lead to antibody production, was also impaired. Thus, it appears that short-lived T lymphocytes control the maturation of macrophages up to a stage at which they can present antigen-specific T cells with antigen in an immunogenic form. We found that such T lymphocytes also control the phagocytic properties of macrophages, yet the impairment of their immunogenic properties does not seem to be derived from decreased phagocytosis.
Crystal structure of galactan-binding mouse immunoglobulin J539 Fab at 4.5-A resolution.
Authors: Navia M A , Segal D M , Padlan E A , Davies D R , Rao N , Rudikoff S , Potter M
Published: May 2006

An electron-density map of the mouse galactan-binding immunoglobulin J539 (IgA2,kappa) Fab has been calculated to a resolution of 4.5 A by the method of heavy atom isomorphous replacement with four derivatives. The map has been interpreted with the aid of a computer program which systematically searched for the best fit between the electron-density map and the known coordinates of individual immunoglobulin domains. The quaternary structure of J539 Fab at this resolution appears similar to that of another mouse immunoglobulin, IgA2,kappa Fab, McPC603. The model coordinates for J539 Fab should allow us to proceed directly to a high-resolution structure determination without further heavy atom isomorphous replacement.
Splicing as a requirement for biogenesis of functional 16S mRNA of simian virus 40.
Authors: Gruss P , Lai C J , Dhar R , Khoury G
Published: May 2006

Simian virus 40 deletion mutants were constructed lacking specifically the intervening sequences for a late viral mRNA. The construction method involved the replacement of portions of the late simian virus 40 genes with the DNA segment from reverse transcription of the viral mRNAs. Restriction endonuclease cleavage and sequence analysis confirmed the precise structure of the mutant DNAs and demonstrated that they contained the genetic information for VP1, including all potential 5' ends for the late viral RNAs. Thus, the primary late transcription product(s) of this mutant should have the structure of functional 16S mRNAs. Complementation analysis as well as immunoprecipitation showed, however, that deletion of the intervening sequences from this mutant prevented the expression of VP1. The nature of this failure appears to be a defect in the posttranscriptional processing of the viral RNA. These results indicate that splicing is an essential function in the biogenesis of certain mRNAs.
Eukaryotic mRNA cap binding protein: purification by affinity chromatography on sepharose-coupled m7GDP.
Authors: Sonenberg N , Rupprecht K M , Hecht S M , Shatkin A J
Published: May 2006

A 24,000-dalton polypeptide that binds strongly and can be specifically crosslinked to the 5'-terminal cap structure m7GpppN in eukaryotic mRNAs has been detected in protein synthesis initiation factor preparations [Proc. Natl. Acad. Sci. USA (1978) 75, 4843--4847]. This polypeptide has been purified to apparent homogeneity by one chromatographic passage through an affinity resin prepared by coupling the levulinic acid O2',3'-acetal of m7GDP to AH-Sepharose 4B. Translation, in HeLa cell extracts, of capped mRNAs including Sindbis virus, reovirus, and rabbit globin mRNAs was stimulated by the cap-binding protein under conditions that did not increase translation of noncapped RNAs of encephalomyocarditis virus and satellite tobacco necrosis virus.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Authors: Towbin H , Staehelin T , Gordon J
Published: May 2006

A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
Photooxidation of the high-potential iron—sulfur center in chloroplasts
Authors: Whitmarsh John , Cramer William A.
Published: May 2006

The high-potential iron—sulfur center associated with the
g
= 1.89-1.90 electron paramagnetic resonance signal in spinach chloroplasts is fully reduced in the dark. It is photooxidized in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and reduced after illumination with red actinic light in the absence of inhibitors. The light-induced redox changes of the high-potential iron—sulfur center are shown to correlate with those of plastocyanin detected at
g
= 2.05. Demonstration of light-induced redox changes in the high-potential iron—sulfur center provides support for a functional role of this component in the chloroplast electron transport chain.
Synthesis of labile, serum-dependent protein in early G
1
controls animal cell growth
Authors: Rossow Peter W. , Riddle Veronica G. H. , Pardee Arthur B.
Published: May 2006

We present a model to account for several major observations on growth control of animal cells in culture. This model is tested by means of kinetic experiments which show that exponentially growing animal cells whose ability to synthesize total protein has been inhibited with cycloheximide (by up to 70%) grow at rates approximately proportional to their rates of protein synthesis. However, virtually the entire elongation of the cell cycle occurs in the part of the G
1
phase that depends on a high concentration of serum in the medium. This part of the cycle has earlier been suggested to lie prior to the restriction point—i.e., the point beyond the main regulatory processes of G
1
. The remainder of the cycle, from restriction point to mitosis, is markedly insensitive to these concentrations of cycloheximide as well as to growth regulation. We quantitatively account for the specific lengthening of that part of the cycle involved in growth regulation by assuming that cells must accumulate a specific protein in a critical amount before they can proceed beyond the restriction point. The lability of this protein (half-life about 2 hr) makes its accumulation unusually sensitive to inhibition of total protein synthesis by cycloheximide. Its production appears to depend on growth factors provided by serum. The model can also account for greater variations of G
1
durations as the growth of cell populations is made slower. It also predicts two sorts of quiescence: one of cells slowly traversing G
1
, in slightly suboptimal conditions; the other of cells that enter G
0
under inadequate conditions. Transformation of different sorts could create cells with altered variables for initiation, synthesis, or inactivation of the regulatory protein or could altogether eliminate the need for the protein.
Differentiation of 3T3-L2 fibroblasts into adipose cells in bromodeoxyuridine-suppressed cultures.
Authors: Russell T R
Published: May 2006

Growth of 3T3-L2 fibroblasts in medium containing 5-bromo-2'-deoxyuridine prevents the spontaneous cytodifferentiation of these cells into adipose cells. Treatment of bromodeoxyuridine-suppressed cultures at confluence with 1-methyl-3-isobutylxanthine, a promoter of the adipose conversion, results in differentiation of the fibroblasts into adipose cells but the extent of the conversion is less than that seen in cultures not treated with bromodeoxyuridine. 1-Methyl-3-isobutylxanthine appears to function without altering the amount of bromodeoxyuridine present in DNA.
Presence and expression of Friend erythroleukemia virus-related sequences in normal and leukemic mouse tissues
Authors: Bernstein Alan , Gamble Catherine , Penrose Donna , Mak Tak W.
Published: May 2006

The nature and distribution of sequences related to the murine erythroleukemia virus, Friend spleen focus-forming virus (SFFV), have been analyzed by using a radioactive cDNA probe specific for the SFFV genome (cDNA
sff
). From the proportion of high molecular weight viral [
32
P]RNA which hybridized to cDNA
sff
, it was estimated that these sequences represent about 50% of the SFFV genome, indicating a genetic complexity of about 3300 nucleotides. cDNA
sff
hybridized extensively (80-95%) to SFFV virion RNA and to cellular RNA from murine and rat cells productively or nonproductively infected with SFFV. Only background homology was detected between cDNA
sff
and viral RNA from a number of murine [Friend murine leukemia virus (MuLV), Moloney-MuLV, and Kirsten sarcoma virus] and nonmurine (Rous sarcoma virus, feline leukemia virus, baboon endogenous virus, and Mason—Pfizer mammary tumor virus) retroviruses. Limited homology was also detected to a number of murine xenotropic and mink cell focus-inducing viruses (20-35%) as well as Rauscher leukemia virus (50%). Nucleotide sequences homologous to cDNA
sff
were also detected in the DNA of normal cells of several mouse strains as single or a few copies per cell. Thermal denaturation analysis indicated that duplexes formed between cDNA
sff
and normal DBA/2J cellular DNA have a reduction in melting temperature of 2°C when compared with the dissociation of hybrids between cDNA
sff
and homologous sequences in SFFV-infected mouse spleen cell DNA. Examination of cellular RNA from uninfected mouse cells indicated that SFFV-related RNA sequences were also expressed in varying degrees in different tissues of adult DBA/2J mice. The highest amounts were observed in cells from bone marrow and spleen, whereas considerably lower amounts were found in cells from the thymus and kidney. No SFFV-related sequences could be detected in RNA extracted from liver, muscle, or fibroblasts. The presence of these SFFV-related sequences in normal, uninfected mouse cell DNA and their differential expression in hematopoietic tissues suggest that these sequences may be an integral part of the program of both normal and leukemic hematopoietic cell differentiation.
Fluorescent phallotoxin, a tool for the visualization of cellular actin.
Authors: Wulf E , Deboben A , Bautz F A , Faulstich H , Wieland T
Published: May 2006

A fluorescent derivative of phalloidin has been synthesized possessing high affinity to filamentous actin. This compound was used for visualization of actin-containing structures in eukaryotic nonmuscle cells. Due to its low molecular weight (1250), fixation for formaldehyde was sufficient to render the membrane permeable for the labeled peptide. Bundles of microfilaments are the predominant pattern in the flat rat kangaroo PtK1 cells, whereas a net of concentric fibers characterizes the more spherical bovine kidney MDBK cells. Specificity of staining was confirmed by competition experiments with unlabeled phalloidin.
Studies on murine Ss protein: demonstration that S region encodes structural gene for fourth component of complement.
Authors: Carroll M C , Capra J D
Published: May 2006

Although genes controlling the expression of certain complement components have been shown to be linked to the major histocompatibility complex of several species, the structural genes that encode these molecules have been more difficult to map. In this study, the three constitutive polypeptide chains of the fourth component of murine complement (C4) (alpha, beta, and gamma) were isolated from 14 different inbred strains and compared by peptide mapping on analytical sodium dodecyl sulfate gels. The peptide patterns of the alpha and gamma subunits appeared to be nearly identical, but two distinctly different patterns were observed for the C4 beta chain. This structural variant was mapped to the S or G region and, as such, provides direct evidence that a structural gene for a complement component is encoded within a major histocompatibility complex.