bluuebunny/crossref_metadata_2025 huggingface dataset:
Steady-state kinetic study of fatty acid synthase from chicken liver.
Authors: Cox B G , Hammes G G
Published: May 2006

The steady-state kinetics of chicken liver fatty acid synthase has been studied over the pH range 5.9-8.6 in 0.1 M potassium phosphate/1 mM EDTA at 25.0 degrees C. The steady-state initial velocity, v, which was determined by measuring the rate of consumption of NADPH spectrophotometrically over a wide range of substrate concentrations, followed the rate law v = (formula; see text), in which Ac-CoA is acetyl-CoA, Mal-CoA is malonyl-CoA, the Kj are Michaelis constants, the Kj,i are inhibition constants, kcat is the turnover number, and [E0] is the total enzyme concentration. The product CoA is an inhibitor at high concentrations but activates the enzyme at low concentrations when the concentration of Ac-CoA is high. The rate law can be derived from a simple multistep mechanism; in terms of this mechanism, the Michaelis constants are lower bounds to the substrate dissociation constants, and the turnover number contains the first-order rate constants characterizing the reactions required to produce palmitic acid. Plots of kcat, kcat/KN, kcat/KA, and kcat/KM versus pH are bell shaped. Analysis of the results in terms of two ionizable groups indicates that in all cases an ionizable group with an apparent pKa of approximately equal to 6 is of importance. For kcat and kcat/KN, the apparent pKa of the second ionizable group is approximately equal to 7.8, whereas for kcat/KA and kcat/KM, it is approximately equal to 7.
New view of lipid bilayer dynamics from 2H and 13C NMR relaxation time measurements.
Authors: Brown M F , Ribeiro A A , Williams G D
Published: May 2006

Natural abundance 13C spin-lattice (T1) relaxation time measurements are reported for unilamellar vesicles of 1,2-dipalmitoylphosphatidylcholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), in the liquid crystalline phase, at magnetic field strengths of 1.40, 1.87, 2.35, 4.23, 7.05, 8.45, and 11.7 tesla (resonance frequencies of 15.0, 20.0, 25.1, 45.3, 75.5, 90.5, and 126 MHz, respectively), and the results are compared to previous 2H T1 studies of multilamellar dispersions. For both the 13C and 2H T1 studies, a dramatic frequency dependence of the relaxation was observed. At superconducting magnetic field strengths (4.23-11.7 tesla), plots of the 13C T1(-1) relaxation rates as a function of acyl chain segment position clearly reveal the characteristic "plateau" signature of the liquid crystalline phase, as found previously from 2H NMR studies. The dependence of T1(1) on ordering, determined previously from 2H NMR, and the T1(-1) dependence on frequency, determined from both 13C and 2H NMR studies, suggest that a unified picture of the bilayer molecular dynamics can be provided by a simple relaxation law of the form T1(-1) approximately equal to A tau f + BS2C-H omega -1/2(0). In the above expression, A and B are constants, SC-H (= SC-D) is the bond segmental order parameter, and omega 0 is the nuclear Larmor frequency. The first (A) term includes contributions from fast, local segmental motions characterized by the effective correlation time tau f, whereas the second (B) term describes slower, collective fluctuations in the local ordering. The value of tau f approximately equal to 10(-11) sec, obtained by extrapolating T1(-1) to infinite frequency, suggests that the segmental microviscosity of the bilayer hydrocarbon region does not differ appreciably from that of the equivalent n-paraffinic liquids of similar chain length.
Autoimmune response directed against conserved determinants of nuclear envelope proteins in a patient with linear scleroderma.
Authors: McKeon F D , Tuffanelli D L , Fukuyama K , Kirschner M W
Published: May 2006

We have studied the autoantibodies in the serum of a patient with linear scleroderma that specifically recognize the nuclear envelope of cultured cells. These antibodies bind to conserved determinants of nuclear lamins, the predominant mammalian nuclear envelope proteins. Of the three mammalian nuclear lamin proteins (970, P68, and P60), only P70 and P60 bind the autoantibodies. In addition, two proteins of the Drosophila embryonic nuclear matrix, P70 and P68, bind these autoantibodies. We have used nuclear matrices to isolate the autoantibodies from the patient's serum that react to the nuclear lamins. At least three different IgG heavy chains were found to be involved in this autoimmune response to nuclear lamins, indicating that this response is not due to the expansion of a single B-cell clone.
Trans-acting temporal locus within the beta-glucuronidase gene complex.
Authors: Lusis A J , Chapman V M , Wangenstein R W , Paigen K
Published: May 2006

Mice carrying the [Gus]H haplotype of the beta-glucuronidase gene complex have considerably decreased enzyme levels and a decreased rate of enzyme synthesis. This is now shown to result from the action of two regulatory loci within the gene complex. One is a systemic regulator, Gus-u, that acts cis to cause a uniform reduction in enzyme levels in all tissues. The other is a temporal locus, Gus-t, that acts trans to cause abrupt switches in the rate of enzyme synthesis in only certain tissues and at characteristic stages of development. The distinction between these two loci was made possible by the introduction of a method for quantitating the relative numbers of A and H allozyme subunits in beta-glucuronidase tetramers. The procedure involves purification of the enzyme, cleavage at methionyl residues with CNBr, isoelectric focusing to separate the peptides, and quantitation of the peptide containing the A/H amino acid substitution. The presence of a trans-acting regulatory locus within a gene complex raises evolutionary and functional questions about why it is located there and how it acts.
Nickel-sequestering renal glycoprotein.
Authors: Abdulwajid A W , Sarkar B
Published: May 2006

Kidney is the target organ where nickel is accumulated and subsequently excreted in the urine after an intraperitoneal administration of 63NiCl2 in rats. The radioactive nickel is found mostly bound to a low molecular weight protein in kidney, which was isolated, purified, and partially characterized in this study. Homogeneity of this protein was determined by polyacrylamide gel electrophoresis. Amino acid analysis showed the presence of high amounts of glycine and proline and low amounts of phenylalanine, tyrosine, hydroxyproline, and hydroxylysine. The protein was found to be a glycoprotein with a carbohydrate content of 10% (wt/wt). Preliminary carbohydrate analysis showed that this glycoprotein is a high mannose-type containing mannose, galactose/glucose, and glucosamine. On the basis of the amino acid and carbohydrate analyses, the molecular weight of the glycoprotein is about 15,000-16,000. In vitro addition of nickel to the kidney cytosol also showed the presence of this protein. The protein appeared not to be affected or altered in its nickel-binding capacity by intraperitoneal or intravenous administration of actinomycin D, indicating it to be a noninducible protein. The glycoprotein demonstrated many characteristics of renal basement membrane. It is proposed that this protein is either a part of the renal basement membrane or is a part of the procollagen in the process of its conversion to collagen of the renal basement protein. The protein has a high affinity for nickel. It also may possess a similar binding affinity for other metals as well and may constitute a natural process of handling toxic levels of metals to be excreted.
Localization of prostaglandin D2 binding protein and NADP-linked 15-hydroxyprostaglandin D2 dehydrogenase in the Purkinje cells of miniature pig cerebellum.
Authors: Watanabe Y , Yamashita A , Tokumoto H , Hayaishi O
Published: May 2006

High-affinity binding sites specific for prostaglandin D2 were visualized by the autoradiographical technique assisted by computerized image processing and color coding. In the cerebellum of miniature pig, high density of the binding was observed in the cortex, mainly in the Purkinje cell layer. Immunohistochemical studies on the cellular localization of NADP-linked 15-hydroxyprostaglandin D2 dehydrogenase were performed with the same cerebellum and revealed the presence of this enzyme also in the Purkinje cells. These findings imply that prostaglandin D2 binds to the Purkinje cells and, after its action, is degraded in situ by the specific degradative enzyme.
Primary structures of human alpha-fetoprotein and its mRNA.
Authors: Morinaga T , Sakai M , Wegmann T G , Tamaoki T
Published: May 2006

DNA complementary to human alpha-fetoprotein (AFP)mRNA was cloned in the plasmid pBR322. Analysis of three overlapping cDNA clones revealed most of the nucleotide sequence of AFP mRNA, and the remaining nucleotides at the 5' end of the mRNA were elucidated from a cloned genomic DNA fragment. The amino acid sequence was deduced from the nucleotide sequence, which revealed 19 amino acids in the signal sequence and 590 amino acids in mature AFP. There are 15 regularly spaced disulfide bridges, which generate a folding structure having three repeating domains. There is one potential N-glycosylation site, Asn-Phe-Thr, in the amino acid sequence. In comparison with mouse AFP, 66% of the amino acid sequence was conserved, with the highest identity (72%) in domain 3, followed by domain 2 (67%) and domain 1 (59%). In comparison with human albumin, a 39% conservation of primary structure was found. Again, the similarity was the highest in domain 3 and the lowest in domain 1. Human AFP and human albumin are similar in overall structure, but certain parts of the molecules differ significantly in their predicted secondary structure.
Envelope gene of the Friend spleen focus-forming virus: deletion and insertions in 3' gp70/p15E-encoding region have resulted in unique features in the primary structure of its protein product.
Authors: Wolff L , Scolnick E , Ruscetti S
Published: May 2006

A nucleotide sequence was determined for the envelope (env) gene of the polycythemia-inducing strain of the acute leukemia-inducing Friend spleen focus-forming virus (SFFV) and from this the amino acid sequence of its gene product, gp52, was deduced. All major elements of the gene were found to be related to genes of other retroviruses that code for functional glycoproteins. Although the carboxyl terminus of gp52 is encoded by sequences highly related to sequences in its putative parent, ecotropic Friend murine leukemia virus, the majority of the protein (69%), including the amino terminus, is encoded by dualtropic virus-like sequences. Nucleotide sequence comparisons suggest that the nonecotropic region may be more closely related to the 5' substitution in dualtropic mink cell focus-inducing viruses that it is to the 5' end of xenotropic virus env genes. A large deletion and two unique insertions have been located in the env gene of polycythemia-inducing SFFV and may account for some of the unusual structural characteristics, aberrant processing, and pathogenic properties of gp52. As a consequence of the deletion, amino-terminal gp70 and carboxyl-terminal p15E-encoding sequences are juxtaposed and it appears that translation from the p15E region, 3' to the deletion, continues in the standard reading frame used by other retroviruses. Insertions of six base pairs and one base pair at the very 3' end of the gp52-encoding region results in a SFFV-unique amino acid sequence and a premature termination codon.
Ornitholimnology: Effects of grazing by the Andean flamingo (
Phoenicoparrus andinus
)
Authors: Hurlbert Stuart H. , Chang Cecily C. Y.
Published: May 2006

Experimental exclusion of the Andean flamingo (
Phoenicoparrus andinus
) from shallow water areas of a salt lake in the Bolivian Andes caused large increases in the biomass of microorganisms inhabiting the surface sediments, especially a large diatom (
Surirella wetzeli
), amebas, ciliates, and nematodes. This is a conservative demonstration of the influences that water birds in general exert on the structure of aquatic ecosystems.
Three organizations of human DNA.
Authors: Strayer D , Heintz N , Roeder R , Gillespie D
Published: May 2006

Human DNA was denatured, annealed to low C0t, and fractionated by centrifugation to equilibrium on gradients of NaI. Three well-defined zones resulted. The topmost, least dense zone contained satellite DNA. The bottom zone contained molecules bearing infrequent sequences and short repeated DNA. The middle zone contained molecules bearing infrequent sequences, short repeated DNA, and long repeated DNA. The NaI pattern was independent of single-strand DNA chain length from 5 to 30 kilobases. Structural genes were found in the organization bearing long repeats.
Defective myoblasts identified in Duchenne muscular dystrophy.
Authors: Blau H M , Webster C , Pavlath G K
Published: May 2006

A defect in the proliferative capacity of satellite cells, mononucleated precursors of mature muscle fibers, was found in clonal analyses of cells cultured from Duchenne muscular dystrophy (DMD) patients. The total yield of myoblasts per gram of muscle biopsy was decreased to 5% of normal. Of the DMD myoblast clones obtained, a large proportion contained a morphological class of flat distended cells that had an increased generation time and ceased to proliferate beyond 100-1,000 cells but could be induced to fuse and form myotubes. The altered muscle phenotype was detected in all cultures from DMD patients but was rarely found among myoblasts of controls. By age 14 yr, it comprised as man as 90% of DMD myoblasts. The remaining DMD myoblast clones, which initially grew well, had severely impaired proliferative capacity upon passage and further cultivation. Eventually all myoblasts from DMD muscle tissue exhibited defective growth potential. In contrast, the fibroblast yield and proliferative capacity from DMD samples did not differ from normal. Based on these findings, we propose a hypothesis for the etiology of DMD: Dividing myoblasts are required for muscle growth and maintenance, and the limited capacity of DMD myoblasts to grow is directly related to the progressive muscle degeneration characteristic of the disease.
Escherichia coli polymerase I can use O2-methyldeoxythymidine or O4-methyldeoxythymidine in place of deoxythymidine in primed poly(dA-dT).poly(dA-dT) synthesis.
Authors: Singer B , Sági J , Kuśmierek J T
Published: May 2006

O2-and O4-alkyldeoxythymidine are among the four O-alkyl base-modified derivatives produced by the reaction of N-nitroso alkylating agents with nucleic acids in vitro and in vivo. We find that both O2- and O4-methyl-dTTP can substitute for dTTP in alternating poly(dA-dT)-primed DNA synthesis. Up to 22% of the pyrimidines in the newly synthesized polymer were found by HPLC analysis to be O-methyldeoxythymidine. Little polymer synthesis was observed in the absence of dTTP. However, the O-methyl-dTTPs did not inhibit polymerization of dATP and dTTP. Polymers containing O2- or O4-methyldeoxythymidine were obtained in good yield, retaining the secondary structure of alternating poly(dA-dT). This was shown by the data for thermal transition under different conditions. In contrast, poly(dA-dT).poly(dA-dT) methylated or ethylated to less than 4% total modification by alkylnitrosoureas had a distinctly less stable structure. Neither O2- nor O4-methyldeoxythymidine can form more than one hydrogen bond with adenosine. The unchanged secondary structure of polymers containing these modified thymidines indicates that stacking interactions must play a major role in helix stabilization. O-Alkyldeoxythymidine may be formed by N-nitroso carcinogens that react intracellularly. We have shown that the triphosphates can be utilized by Escherichia coli DNA polymerase I as dTTP. The incorporated O4-methyl-dT causes misincorporation of G, both in transcription and synthesis. When O2-methyl-dT is present, less, but definite, misincorporation results.
Recombination between antibody heavy chain variable-region genes: evidence for gene conversion.
Authors: Krawinkel U , Zoebelein G , Brüggemann M , Radbruch A , Rajewsky K
Published: May 2006

The murine hybridoma line B1-8.delta 1 secretes monoclonal IgD lambda 1 antibodies specific for the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP). The variable (V) region of these antibodies is defined by a characteristic pattern of idiotopes. A spontaneous V-region variant (B1-8.V1) with altered idiotope pattern was selected. The structural variation is confined to the V region of the heavy chain. It was shown previously that the variant V region is encoded by a gene that was generated by a crossover between the rearranged VDJ gene of the wild type (B1-8.delta 1) and a neighboring germ-line VH gene. In the present study the nucleotide sequence of coding and flanking regions of the VH gene expressed in variant B1-8.V1 was determined. Wild-type and variant VH genes differ at 15 positions in a region between leader sequence and codon 66. The sequence of the region carrying the substitutions is identical to the sequence of the corresponding region in a neighboring germ-line VH gene. This implies that the variant VH gene was generated by a mechanism of recombination more complicated than single crossover. Gene conversion as the mechanism of the recombination is discussed.
Activation of plant quinate:NAD
+
3-oxidoreductase by Ca
2+
and calmodulin
Authors: Ranjeva Raoul , Refeno Germain , Boudet Alain M. , Marmé Dieter
Published: May 2006

Quinate:NAD
+
3-oxidoreductase (EC 1.1.1.24) from carrot cell suspension cultures has previously been shown to be activated by phosphorylation and inactivated by dephosphorylation. Here it is shown that the reactivation of the inactivated quinate:NAD
+
oxidoreductase is an enzyme-mediated process that requires ATP and protein kinase activity. The reactivation is completely inhibited by EGTA and can be restored by the addition of Ca
2+
. Cyclic AMP at concentrations up to 5 μM did not have any effect on the reactivation either with or without EGTA in the medium. Calmodulin-depleted fractions containing quinate:NAD
+
oxidoreductase were obtained by passage of the crude extracts through an affinity column of 2-chloro-10-(3-aminopropyl)phenothiazine coupled to Sepharose 4B. The enzyme in this calmodulin-deficient fraction could be inactivated but not reactivated even in the presence of ATP and Ca
2+
. However, addition of bovine brain calmodulin completely restored the activity of the enzyme. Half-maximal activation occurred at 130 nM calmodulin. We conclude from these data that the quinate:NAD
+
oxidoreductase is activated by a Ca
2+
- and calmodulin-dependent plant protein kinase.
Sequence coding for the alphavirus nonstructural proteins is interrupted by an opal termination codon.
Authors: Strauss E G , Rice C M , Strauss J H
Published: May 2006

We have obtained the nucleotide sequence of the genomic RNAs of two alphaviruses, Sindbis virus and Middelburg virus, over an extensive region encoding the nonstructural (replicase) proteins. In both viruses in an equivalent position an opal (UGA) termination codon punctuates a long otherwise open reading frame. The nonstructural proteins are translated as polyprotein precursors that are processed by posttranslational cleavage into four polypeptide chains; the sequence data presented here indicate that the COOH-terminal polypeptide, ns72, may be produced by read-through of this opal codon. The high degree of amino acid homology between the ns72 polypeptides of the two viruses, in contrast to the lack of conserved sequence upstream from the read-through site, suggests that ns72 plays an important role in viral replication, possibly modulating the action of other replicase components.
Phosphorylation of human growth hormone by the epidermal growth factor-stimulated tyrosine kinase.
Authors: Baldwin G S , Grego B , Hearn M T , Knesel J A , Morgan F J , Simpson R J
Published: May 2006

In the present study, we have demonstrated that human growth hormone (hGH) can be phosphorylated by the epidermal growth factor (EGF)-stimulated tyrosine kinase of A431 cell membranes. Phosphotyrosine was the predominant phosphoamino acid released from phosphorylated hGH on partial acid hydrolysis. All five tyrosine-containing tryptic peptides of hGH are also phosphorylated by the EGF-stimulated tyrosine kinase. The highest phosphate incorporation was found for peptide T4 (residues 20-38), which is distinguished by a high frequency of acidic amino acids. The phosphorylated peptides have been characterized by HPLC and two-dimensional mapping on paper. Comparison with the labeled peptides obtained on tryptic digestion of phosphorylated hGH suggests that tyrosine phosphorylation is restricted to two tryptic peptides, T4 (tyrosine-28 or -35) and T6 (tyrosine-42). It is suggested that the absence of early insulin-like activity in the naturally occurring Mr 20,000 variant of hGH, which has an internal deletion spanning residues 32-46, may be a consequence of the loss of the tyrosine phosphorylation sites at residues 35 and 42.
Natural 25,26-dihydroxyvitamin D3 is an epimeric mixture.
Authors: Ikekawa N , Koizumi N , Ohshima E , Ishizuka S , Takeshita T , Tanaka Y , DeLuca H F
Published: May 2006

Radiolabeled 25,26-dihydroxyvitamin D3 was prepared in vitro by using chicken kidney homogenates and in vivo in rats from [23,24-3H]-25-hydroxyvitamin D3. These compounds were mixed with synthetic (25S)- and (25R)-25,26-dihydroxyvitamin D3, converted to the corresponding (+)-alpha-methoxy-alpha-trifluoromethylphenylacetyl esters, and subjected to high-performance liquid chromatography that separates the derivatized epimers. The radiolabeled 25,26-dihydroxyvitamin D3 derivatives were a 1:1 mixture of the 25S and 25R isomers. Similarly unlabeled 25,26-dihydroxyvitamin D3 isolated from the plasma of rats given large amounts of vitamin D3 was shown to be a 1:1 mixture of the S and R isomers. Therefore, naturally occurring 25,26-dihydroxyvitamin D3 is a mixture of the 25R and 25S isomers and not just the S isomer reported previously.
Structural dynamics of human deoxyhemoglobin and hemochrome investigated by nuclear gamma resonance absorption (Mössbauer) spectroscopy.
Authors: Mayo K H , Kucheida D , Parak F , Chien J C
Published: May 2006

Mössbauer spectra of human deoxyhemoglobin and hemochrome frozen solutions, selectively enriched with 57Fe in either the alpha chains or the beta chains, were measured from 4.2 to 250 K. The Lamb-Mössbauer factor, ln f = 4II2 [x2]gamma/lambda 2, was calculated from these spectra and gives the structural dynamics of the iron atom (heme). Large differences in the mean-squared displacement, [x2]gamma, of the heme between hemochrome and deoxyhemoglobin at temperatures above 210 K were observed, demonstrating that when iron is bound to a rigid part of the protein (i.e., distal histidine in hemochrome), motions of the heme are suppressed (i.e., the dynamics are decreased). Comparison of the motions of these two hemoglobins proves that molecular diffusion can be neglected in an analysis of the dynamics below approximately 250 K.
Local anesthetic-membrane interaction: a multiequilibrium model.
Authors: Wang H H , Earnest J , Limbacher H P
Published: May 2006

We report the detection of electrostatic interactions between local anesthetics and membrane phospholipids and proteins. A spin-labeled local anesthetic was used to study how membrane-bound tertiary amine anesthetics interact with major molecular components in the membrane. The nitroxyl reporter group of this spin label is located at the polar end of the amphiphilic local anesthetic; it is therefore a uniquely suitable probe for detecting immobilization of the anesthetic due to binding interactions at the polar regions of the bilayer. The binding properties of this spin-labeled anesthetic to human erythrocyte membranes and to vesicles made from human erythrocyte lipids were studied. Lipid vesicle-bound spin labels give rise to a composite electron spin resonance spectrum from which two subcomponent spectra were resolved. Both components are membrane-bound; the first component has a narrower linewidth, indicating a greater mobility of the nitroxyl moiety of the anesthetic probe. The second component has a broader linewidth, indicating a population of constrained spin labels. We infer from the experimental results that electrostatic binding between cationic anesthetics and anionic phosphate of phospholipids produced the constrained component. In similar studies using erythrocyte ghost membranes, both a mobile (nonelectrostatic) component and a constrained (electrostatic) component were resolved from the composite spectrum. However, the constrained component in this case is much broader than the corresponding constrained component from the vesicles. We interpret this broad component in the erythrocyte membrane as an electrostatic interaction of cationic anesthetic probes with phospholipids and with membrane proteins. We conclude that membrane-bound tertiary amine anesthetics in cationic form do interact selectively with phospholipids and proteins.
Construction of live vaccines by using genetically engineered poxviruses: biological activity of recombinant vaccinia virus expressing influenza virus hemagglutinin.
Authors: Panicali D , Davis S W , Weinberg R L , Paoletti E
Published: May 2006

Recombinant vaccinia viruses containing the cloned hemagglutinin (HA) gene from influenza virus were constructed. The biological activity of these poxvirus vectors was demonstrated both in vitro and in vivo. Expression of HA in cells infected with recombinant vaccinia was detected by using specific anti-HA antiserum and 125I-labeled protein A, showing that HA synthesized under the regulation of vaccinia virus was antigenic. Immunization of rabbits with these recombinant poxviruses resulted in the production of antibodies reactive with authentic influenza HA as detected by radioimmunoassay, by inhibition of HA erythrocyte agglutination, and by neutralization of influenza virus infectivity. The production of antibodies directed against influenza HA suggested that the HA gene expressed in vaccinia is immunogenic. These data indicate the potential of genetically engineered poxviruses for use as generic live vaccine vehicles that have both human and veterinary applications.
Specific high-affinity binding and biologic action of retinoic acid in human neuroblastoma cell lines.
Authors: Haussler M , Sidell N , Kelly M , Donaldson C , Altman A , Mangelsdorf D
Published: May 2006

Neuroblastoma cells are a good model for neuronal development because of their ability to extend neurites in response to various stimuli, including retinoic acid. In the present experiments, we have examined five human neuroblastoma cell lines (LA-N-1, IMR-32, LA-N-5, SK-N-MC, and CHP-100) for the presence of cellular retinoic acid binding protein (CRABP), a receptor-like protein implicated in the molecular functioning of vitamin A. CRABP is identified and quantitated by sucrose gradient centrifugation, selective inhibition by the mercurial reagent p-chloromercuribenzene sulfonic acid (PCMBS), and saturation analysis. All five lines contain significant levels of cytosolic CRABP (2.5-7.5 pmol/mg of protein), which display typical properties of specific high affinity retinoic acid binding, a sedimentation coefficient of 2 S, and inhibition by PCMBS. Three of the lines (LA-N-1, IMR-32, and LA-N-5) are strongly growth inhibited by 1 microM retinoic acid in monolayer culture, whereas two (LA-N-1 and LA-N-5) undergo marked differentiation to a stellate, fusiform morphology with characteristic neurite outgrowths. The SK-N-MC and CHP-100 lines are relatively resistant to the antiproliferative effects of retinoic acid under these conditions. Nevertheless, all five lines are effectively inhibited by retinoic acid in their ability to form anchorage-independent colonies in soft agar. Thus, although CRABP is not necessarily correlated with growth inhibition in monolayer culture, it is associated with retinoic acid's ability to inhibit neuroblastoma colony formation in soft agar. More experiments will be required to determine if this effect on growth in soft agar reflects the putative ability of retinoic acid to convert tumorigenic neuroblastoma cell lines into the normal differentiated phenotype.
Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine.
Authors: Dolbeare F , Gratzner H , Pallavicini M G , Gray J W
Published: May 2006

We have developed a procedure for simultaneous flow cytometric measurement of cellular DNA content and amount of BrdUrd incorporated into cellular DNA. Propidium iodide was used as a fluorescent probe for total cellular DNA and a monoclonal antibody against BrdUrd was used as a probe for BrdUrd incorporated into DNA. Fluorescein-labeled goat anti-mouse antibody was used to fluorescently label the bound anti-BrdUrd probe. Bivariate DNA/BrdUrd distributions measured for Chinese hamster ovary cells labeled for 30 min with BrdUrd clearly show the G1-and G2M-phase cells to have low BrdUrd-linked fluorescence and the S-phase cells to have high BrdUrd-linked fluorescence. Cell cycle traverse rates were estimated for Chinese hamster ovary cells from bivariate distributions measured for samples taken periodically after pulse labeling with BrdUrd. Bivariate DNA/BrdUrd distributions were also applied in the analysis of the response of C3H murine bone marrow cells to treatment in vivo with 1-beta-D-arabinofuranosylcytosine (araC). Bivariate distributions were measured for bone marrow cells taken from mice that were pulse labeled with BrdUrd at various times after treatment with araC. The resulting DNA/BrdUrd sequences show the kinetics of recovery from araC and allow discrimination of the araC sterilized cells.
1 alpha,25-dihydroxyvitamin D3 promotes fusion of mouse alveolar macrophages both by a direct mechanism and by a spleen cell-mediated indirect mechanism.
Authors: Abe E , Miyaura C , Tanaka H , Shiina Y , Kuribayashi T , Suda S , Nishii Y , DeLuca H F , Suda T
Published: May 2006

Extensive fusion was induced in mouse alveolar macrophages by treatment with conditioned media obtained from spleen cell cultures treated with 15 micrograms of phytohemagglutinin or concanavalin A per ml or with 12 nM 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]. The fusion rate was 80-90% on day 3. In addition, 1 alpha,25(OH)2D3 added directly to alveolar macrophages induced fusion of about 35% of the cells on day 3, whereas direct addition of phytohemagglutinin and concanavalin A did not enhance fusion at all. When conditioned media from spleen cell or T cell cultures treated with 12 nM 1 alpha,25(OH)2D3 were applied to a Sephadex G-100 column, a fusion factor (Mr 37,000-70,000) could be separated from 1 alpha,25(OH)2D3. 1 alpha,25(OH)2D3 induced fusion at 0.012-120 nM in a dose-dependent manner both by direct action and by spleen cell-mediated indirect action, but the fusion rate was always much greater in the latter than in the former at each concentration of the vitamin. Of the vitamin D3 derivatives tested, 1 alpha,25(OH)2D3 was the most potent, followed successively by 1 alpha,24R,25-trihydroxyvitamin D3, 1 alpha-hydroxyvitamin D3, 25-hydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3. These results clearly indicate that 1 alpha,25(OH)2D3 induces fusion of mouse alveolar macrophages by both a direct and an indirect mechanism, the latter mediated by spleen cells, probably by T cells.
Identification and initial characterization of a rat monoclonal antibody reactive with the murine interleukin 2 receptor-ligand complex.
Authors: Malek T R , Robb R J , Shevach E M
Published: May 2006

Xenogeneic monoclonal antibodies were prepared to the murine interleukin 2 (IL-2)-dependent HT2 cell line. One rat IgM monoclonal antibody (7D4) was identified that inhibited proliferation of the HT2 cells and of IL-2-dependent CTLL cells in the presence of crude rat IL-2 as well as of purified human IL-2. The level of inhibition was dependent on both antibody and IL-2 concentration. Cell distribution studies using a fluorescence-activated cell sorter showed that the antigen identified by 7D4 is expressed at a high density on HT2 cells and on concanavalin A (Con A)-induced T-cell blasts and at a substantially lower density on lipopolysaccharide-induced B-cell blasts; 7D4 binding was not detected on greater than 95% of nonactivated thymocytes, T cells, or B cells. Competition binding studies indicated that 7D4 fails to inhibit the binding of 3H-labeled human IL-2 to CTLL cells. However, 7D4 specifically immunoprecipitated 3H-labeled human IL-2 from detergent extracts of HT2 cells or Con A-induced T-cell blasts that had been pulsed with [3H]IL-2; in contrast, 7D4 did not react with free [3H]IL-2. Initial biochemical analysis of immunoprecipitates with 7D4 of detergent extracts from surface-iodinated Con A-activated spleen cells showed a major band having apparent molecular weight of 48,000-62,000. Collectively, these results suggest that 7D4 detects an epitope on the IL-2 receptor distal to the ligand binding site or another molecule that physically associates with the receptor.
Cholesterol synthesis and low density lipoprotein uptake are regulated independently in rat small intestinal epithelium.
Authors: Stange E F , Dietschy J M
Published: May 2006

We have compared the rates of low density lipoprotein (LDL) uptake and cholesterol synthesis in the rat intestine. By using a constant infusion technique, total and receptor-independent uptake was determined with homologous rat LDL (rLDL) and methylated human LDL (Me-hLDL), respectively. The absolute rates of sterol synthesis were measured with [1-(14) C]-octanoate and [3H]water. The rates of rLDL uptake in whole gut segments were similar along the length of the small intestine, whereas the rates of sterol synthesis varied over a 5-fold range and were highest in the duodenum and distal ileum. When the mucosal epithelium was fractionated along the villus/crypt axis, both rLDL and Me-hLDL clearance by the enterocytes increased approximately 3-fold in going from the upper villus to the crypt cell fractions, in both jejunum and ileum. In both the whole gut segments and isolated cells, approximately 60% of LDL uptake was receptor dependent. When the rates of rLDL cholesterol uptake were calculated and related to the absolute rates of sterol synthesis in the same cell fractions in vivo, both processes were found to be distributed similarly along the villus/crypt axis. Furthermore, the majority of mucosal cholesterol (64-86%) was derived from local synthesis rather than from rLDL uptake at all locations along the intestinal villus. Finally, when sterol synthesis in the epithelial cells was varied up to 7-fold by feeding cholesterol, triglyceride, cholestyramine, or surfomer, rLDL uptake was essentially unchanged. Thus, in intestinal epithelial cells in vivo, the rate of LDL uptake was constant under circumstances in which changing needs for cellular cholesterol were met by changes in the rates of sterol synthesis.
Ultrastructural networks in growth cones and neurites of cultured central nervous system neurons.
Authors: Tsui H C , Ris H , Klein W L
Published: May 2006

We have examined growth cones and neurites of cultured central nervous system neurons by high-voltage electron microscopy. Embryonic chicken retina cells were cultured on polylysine-treated and Formvar-coated gold grids for 2-6 days, fixed, and critical point dried. Growth cones and neurites were examined as unembedded whole mounts. Three-dimensional images from stereo-pair electron micrographs of these regions showed a high degree of ultrastructural articulation, with distinct, non-tapering filaments (5-9 nm in diameter) joining both cytoskeletal and membranous components. In the central regions of growth cones, interconnected structures included microtubules, large membranous sacs (up to 400 nm), and irregular vesicles (25-75 nm). A denser filamentous network was prevalent at the edges of growth cones. This network, which frequently adjoined the surface membrane, linked vesicles of uniform size (35-40 nm). Such vesicles often were seen densely packed in growth cone protrusions that were about the size of small synaptic boutons. Prevalent structural interconnections within growth cones conceivably could play a logistic role in specific membrane assembly, intracellular transport, endocytosis, and secretion. Because such processes are not unique to growth cones, the extensive linkages we have observed may have implications for cytoplasmic structure in general.
Viscous, resistive magnetohydrodynamic stability computed by spectral techniques
Authors: Dahlburg R. B. , Zang T. A. , Montgomery D. , Hussaini M. Y.
Published: May 2006

Expansions in Chebyshev polynomials are used to study the linear stability of one-dimensional magnetohydrodynamic quasiequilibria, in the presence of finite resistivity and viscosity. The method is modeled on the one used by Orszag in accurate computation of solutions of the Orr-Sommerfeld equation. Two Reynolds-like numbers involving Alfvén speeds, length scales, kinematic viscosity, and magnetic diffusivity govern the stability boundaries, which are determined by the
geometric mean
of the two Reynolds-like numbers. Marginal stability curves, growth rates versus Reynolds-like numbers, and growth rates versus parallel wave numbers are exhibited. A numerical result that appears general is that instability has been found to be associated with inflection points in the current profile, though no general analytical proof has emerged. It is possible that nonlinear subcritical three-dimensional instabilities may exist, similar to those in Poiseuille and Couette flow.
Multiple quantum two-dimensional 1H--15N nuclear magnetic resonance spectroscopy: chemical shift correlation maps for exchangeable imino protons of Escherichia coli tRNAMetf in water.
Authors: Griffey R H , Poulter C D , Bax A , Hawkins B L , Yamaizumi Z , Nishimura S
Published: May 2006

A procedure based on multiple quantum two-dimensional nuclear magnetic resonance spectroscopy is described for generation of 1H--15N chemical shift correlation maps. The method is used to obtain 15N chemical shifts for the exchangeable imino protons in 1H--15N units of site-specifically labeled Escherichia coli tRNAMetf in water. The high sensitivity and excellent chemical shift dispersion of the multiple quantum two-dimensional technique make it ideally suited for studying protonated nitrogens by NMR.
Induction of monocytic differentiation and bone resorption by 1,25-dihydroxyvitamin D3.
Authors: Bar-Shavit Z , Teitelbaum S L , Reitsma P , Hall A , Pegg L E , Trial J , Kahn A J
Published: May 2006

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] stimulates bone resorption in man and other vertebrates, in part, by increasing the number of osteoclasts, the principal resorbing cells of bone. Because osteoclasts are very likely derived from a member(s) of the mononuclear phagocyte family, we determined if 1,25(OH)2D3 promotes maturation of these cells by studying its effects on the human promyelocytic leukemia cell line HL-60. Of the vitamin D3 metabolites tested, only 1,25(OH)2D3, at 10(-10) to 10(-7) M, induces the differentiation of HL60 into mono- and multinucleated macrophage-like cells. Phenotypic change is evident within 24 hr and reaches a plateau between 72 and 96 hr of incubation. The changes are metabolite-specific and include (i) adherence to substrate, (ii) acquisition of the morphological features of mature monocytes, (iii) a 4- to 6-fold enhancement in lysozyme synthesis and secretion, (iv) increase in the fraction of alpha-naphthyl acetate esterase-positive cells from approximately 2% to 100% of the population, and (v) the acquisition of several monocyte-associated cell surface antigens. More importantly, treated HL-60 cells acquire the capacity to bind and degrade bone matrix, two of the essential, functional characteristics of osteoclasts and related bone-resorbing cells. These results, considered together with the reported action of 1,25(OH)2D3 on nontransformed mononuclear cells, are consistent with the view that vitamin D3 enhances bone resorption and osteoclastogenesis in vivo by promoting the differentiation of precursor cells.
Connectin: cell surface protein that binds both laminin and actin.
Authors: Brown S S , Malinoff H L , Wicha M S
Published: May 2006

A purified cell surface receptor protein for laminin (Mr = 70,000) isolated from mouse fibrosarcoma cells binds to actin with specificity and high affinity. This binding was demonstrated both by cosedimentation of the receptor with actin and binding of the receptor to actin immobilized on nitrocellulose filters. Specificity was demonstrated by displacement of 35S-labeled receptor by unlabeled receptor. Scatchard analysis of receptor binding to actin yielded a Kd of 6 X 10(-7) M. The receptor was observed to reduce the viscosity of actin filaments. It also caused the formation of bundles of parallel filaments. This observation and the stoichiometry of binding suggest that the receptor binds along the sides of actin filaments. Based on the ability of this receptor to bind both extracellular laminin and intracellular actin, we have named this protein "connectin." Connectin may be an example of a transmembrane protein that is capable of mediating the interaction of a cell with its extracellular matrix.
Human lymphocyte production of immunoreactive thyrotropin.
Authors: Smith E M , Phan M , Kruger T E , Coppenhaver D H , Blalock J E
Published: May 2006

Interferon-alpha inducers were previously shown to cause human lymphocyte production of a corticotropin (ACTH)-like peptide. Thyrotropin (TSH) was not produced under these conditions. In contrast, this report shows that a T-cell mitogen (staphylococcal enterotoxin A), which does not induce the ACTH-like peptide, caused human lymphocyte production of an immunoreactive (ir) TSH. Lymphocyte synthesis of the ir TSH was first detectable at 24 hr, peaked at 48 hr, and thereafter declined. NaDodSO4/polyacrylamide gel electrophoresis of intrinsically radiolabeled lymphocyte-derived ir TSH showed radiolabeled peaks at 80, 50, and 26 kilodaltons. These peaks presumably correspond to trimeric, dimeric, and monomeric TSH-like proteins, respectively. Acid treatment and reduction caused the ir TSH to migrate as a 14-kilodalton peak with a 12-kilodalton shoulder in a gel filtration column run in 6 M guanidine . HCl. Thus, the ir TSH seemed to be composed of subunits with molecular masses corresponding to those of the beta and alpha chains of human TSH, respectively. The ir TSH appeared to be glycoprotein because it bound to a concanavalin A affinity column. Taken together these data suggest that in addition to ACTH, human lymphocytes can also produce a TSH-like substance.
Leukemia cell lines can replace monocytes for mitogen-induced T-lymphocyte responses: this accessory function is dependent upon their differentiation stage.
Authors: Wakasugi H , Harel A , Dokhelar M C , Fradelizi D , Tursz T
Published: May 2006

Highly purified peripheral T lymphocytes do not proliferate in response to phytohemagglutinin A or concanavalin A, unless adherent HLA-DR+ monocytes are added as accessory cells. The accessory function (AF) of monocytes is mediated through the release of interleukin-1 (IL-1). We here report that cells from three human leukemic lines--K562, HL-60, and U-937--could exert AF and efficiently replace monocytes in a 72-hr mitogen-stimulated proliferation assay. This AF was clearly related to precise maturational stages of these cells, since the hematopoietic precursor K562 cells spontaneously exerted high AF but lost this property when treated with differentiation inducers such as sodium butyrate or phorbol 12-myristate 13-acetate (PMA). On the other hand, untreated HL-60 and U-937 cells exhibited no spontaneous AF, but they acquired this function when induced to differentiate either along the granulocytic pathway (dimethyl sulfoxide-treated HL-60 cells) or along the monocytic pathway (PMA-treated HL-60 and U-937 cells). Supernatants from PMA-triggered K562 or HL-60 cells allowed the proliferative response of murine thymocytes to phytohemagglutinin A and were therefore shown to contain IL-1. Analysis of phenotypical markers showed that AF and IL-1 production were not restricted to cells of the monocytic lineage. No HLA-DR antigen could be detected on K562 and HL-60 cells. Thus, the expression of HLA-DR antigens is not required for AF and IL-1 production in response to mitogens. Human leukemia cell lines could provide useful sources of human IL-1.
Genetic mapping of a human class II antigen beta-chain cDNA clone to the SB region of the HLA complex.
Authors: Roux-Dosseto M , Auffray C , Lillie J W , Boss J M , Cohen D , DeMars R , Mawas C , Seidman J G , Strominger J L
Published: May 2006

A class II antigen beta-chain cDNA clone was isolated from a human B-cell cDNA library by using as a probe the murine I-A beta gene. This cDNA clone, pHA beta, was shown to be distinct from the DC beta- and DR beta-related loci by DNA sequence analysis, thus suggesting that it might correspond to a third polymorphic human class II locus, SB, which encodes secondary B-cell antigens. Genetic mapping of this beta-chain cDNA clone to the SB region was performed by the blot hybridization procedure. We showed that (i) within panels of HLA-DR homozygous human B-cell lines and of unrelated individuals who have been typed for HLA antigens, differential mobility of DNA fragments segregated with distinct SB genotypes; (ii) gamma-ray-induced deletion mutants that have lost the expression of DR or DC/MT antigens but maintain SB expression preserved a pattern consistent with (a) their SB phenotype and (b) the genetic independence of the SB locus with respect to DR and DC/MT; and (iii) within an informative family, two siblings differing only for one allele at the SB locus (because of the occurrence of an internal recombination between DR and GLO) and otherwise HLA identical exhibited a restriction enzyme polymorphism linked to the SB locus. Therefore, all available data are compatible with identity between HA beta and SB beta.