bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromosomes in living Escherichia coli cells are segregated into domains of supercoiling.
Authors: Sinden R R , Pettijohn D E
Published: May 2006

Torsional tension in the DNA double helix can be detected in living cells of Escherichia coli from measurements of the rate of trimethylpsoralen photobinding to the intracellular DNA. Here we show that this tension is relaxed in vivo when single-strand DNA breaks are introduced by gamma-irradiation and that approximately 160 nicks per genome equivalent of DNA are required to relax greater than 95% of the tension. Chromosomes containing less than 160 nicks per genome equivalent lose only a part of the tension, depending on the number of nicks. The remaining tension is maintained during incubations of cells at 0 degrees C. Chromosomes with tension relaxed by incubation of cells with inhibitors of DNA gyrase interact with the trimethylpsoralen probe independently of the number of nicks introduced by gamma-irradiation. The results fit a model in which the chromosome in growing E. coli cells (mean generation time, 30 min) is segregated into 43 +/- 10 domains of supercoiling per genome equivalent of DNA or 120 +/- 30 domains per nucleoid. The number of domains is unchanged in cells depleted of nascent RNA by growth with rifampicin, but varies somewhat in cells growing at different rates in different media.
Cytochrome oxidase (a3) heme and copper observed by low-temperature Fourier transform infrared spectroscopy of the CO complex.
Authors: Alben J O , Moh P P , Fiamingo F G , Altschuld R A
Published: May 2006

Carbon monoxide bound to iron or copper in substrate-reduced mitochondrial cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) from beef heart has been used to explore the structural interaction of the a3 heme-copper pocket at 15 K and 80 K in the dark and in the presence of visible light. The vibrational absorptions of CO measured by a Fourier transform infrared interferometer occur in the dark at 1963 cm-1, with small absorptions near 1952 cm-1, and are due to a3 heme--CO complexes. These disappear in strong visible light and are replaced by a major absorption at 2062 cm-1 and a minor one at 2043 cm-1 due to Cu--CO. Relaxation in the dark is rapid and quantitative at 210 K, but becomes negligible below 140 K. The multiple absorptions indicate structural heterogeneity of cytochrome oxidase in mitochondria. The Cu--CO absorptions (vCO) are similar to those in hemocyanin--CO complexes from molluscs (vCO - 2062 cm-1) and crustaceans (vCO = 2043 cm-1). The 2062 cm-1 Cu--CO absorption of cytochrome oxidase is split into two bands at 15 K. Analysis of spectral data suggest the presence of a very nonpolar heme--Cu pocket in which the heme-CO complex is highly ordered, but in which the Cu--CO complex is much more flexible, especially above 80 K. A function for these structures in oxygen reduction is proposed.
Mechanism of tobacco mosaic virus assembly: Incorporation of 4S and 20S protein at pH 7.0 and 20°C
Authors: Shire Steven J. , Stegkert John J. , Schuster Todd M.
Published: May 2006

The mechanism of assembly of tobacco mosaic virus (TMV) has been investigated at pH 7.0 and 20°C by analytical ultracentrifugation. Under these conditions the overall rates of interconversion of 4S and 20S TMV coat protein are sufficiently slow to make possible measurements of the concentrations of remaining 4S and 20S TMV coat protein after addition of homologous RNA to solutions containing, initially, various mass ratios of 20S protein to 4S protein. It has been possible to measure, by schlieren boundary analysis, the relative rates of incorporation of 4S and 20S TMV protein into the growing nucleoprotein rod over the range of initial 20S:4S protein mass ratios from 93:7 to 18:82. The results show that the amount of incorporation of 20S TMV protein depends on the initial 20S:4S mass ratio between ≈100% and 60% 20S protein but that reconstitution can proceed with ≈100% 20S TMV protein to form full virus-size rods. However, when the initial protein solutions have less than 60% 20S protein, ≈80% of the reconstituted nucleoprotein is preferentially formed from 4S coat protein. The remaining ≈20% appears to require preformed 20S coat protein. These results suggest that a larger region of RNA than previously estimated is involved in the rate-limiting nucleation step in assembly and may explain previously conflicting results concerning the elongation phase of assembly when starting with partially assembled rods.
Growth regulation, reverse transformation, and adaptability of 3T3 cells in decreased Mg
2+
concentration
Authors: Rubin Harry
Published: May 2006

A nontransformed and a spontaneously transformed clone of BALB/c 3T3 cells were compared for their capacity to multiply in decreased concentrations of Mg
2+
. Cells of the nontransformed clone were flat, formed regularly patterned, nonoverlapping arrays, required high serum concentration for multiplication, had a low saturation density, and did not make colonies in agar. Cells of the transformed clone were slender and spiky, formed random, overlapping arrays, multiplied in low serum concentrations, and had no fixed saturation density, and 20-30% of them formed colonies in agar. The saturation density of the nontransformed clone was decreased in a growth-limiting supply of Mg
2+
in proportion to the reduction in initial rate of multiplication. At very low Mg
2+
concentrations, saturation occurred when less than half of the surface of the dish was covered with cells. The transformed cells did not reach a stable saturation density in low Mg
2+
concentrations, but their growth rate did slow down when they became crowded, and a transient saturation density was reached at the lowest Mg
2+
concentrations that allowed multiplication. Limiting the supply of Mg
2+
caused the transformed cells to flatten and to assume a regularly patterned, non-overlapping relationship to one another, resembling that of the nontransformed cells. This also occurred in BALB/c 3T3 cells transformed by infection with Moloney mouse sarcoma virus. After 1 week in low concentrations of Mg
2+
, the nontransformed cells began to multiply and to incorporate [
3
H]thymidine at a rapid rate. The transformed cells did so also and, in addition, reverted to their transformed appearance. The intracellular content of Mg
2+
was not significantly decreased when the extracellular concentration was decreased to 1/50th. The results suggest that: (
a
) limited contact among cells already multiplying at a reduced rate is sufficient to halt further multiplication; (
b
) a very small decrease in intracellular Mg
2+
content or in membrane-associated Mg
2+
causes transformed cells to assume aspects of the appearance and behavior of nontransformed cells (i.e., Mg
2+
-regulated reactions may be involved in determining the transformed phenotype); and (
c
) cells multiplying at a slow rate in low concentrations of Mg
2+
begin to multiply faster after about 1 week, due either to an adaptation of the cells or to a change in the cellular microenvironment.
Cooperation of kinetochores and pole in the establishment of monopolar mitotic apparatus.
Authors: Mazia D , Paweletz N , Sluder G , Finze E M
Published: May 2006

Monopolar mitotic apparatus can be produced in sea urchin eggs by a manoeuvre that distributes the four poles of the second mitosis into four separate blastomeres. The pole of the monopolar mitotic apparatus generates a half-spindle that is similar in structural details to the half-spindle of a normal bipolar mitotic apparatus, although the chromosomes are not as well aligned as in a normal metaphase plate. The chromosomes are oriented; one kinetochore faces the pole while its sister kinetochore faces away from the pole. The poleward kinetochore is connected to the pole by bundles of microtubules. No microtubules are seen on the sister kinetochore that faces away from the pole. Therefore, a single pole can direct most of the events in the establishment of a mitotic apparatus. Our interpretation examines the cooperation of kinetochores and poles in the formation of microtubules between them, stressing the half-spindle as the medium of cooperation and leaving open the question whether the kinetochores are origins or terminations of microtubules.
Sequences of the joining region genes for immunoglobulin heavy chains and their role in generation of antibody diversity.
Authors: Gough N M , Bernard O
Published: May 2006

To assess the contribution to immunoglobulin heavy chain diversity made by recombination between variable region (VH) genes and joining region (JH) genes, we have determined the sequence of about 2000 nucleotides spanning the rearranged JH gene cluster associated with the VH gene expressed in plasmacytoma HPC76. The active VH76 gene has recombined with the second germ-line JH gene. The region we have studied contains two other JH genes, designated JH3 and JH4. No other JH gene was found within the region 1000 nucleotides downstream from JH4. Between JH3 and JH4 there is a pseudo-JH sequence with substantial homology to the authentic JH genes. The four JH genes whose sequences now are known can account for all known JH amino acid sequences. The JH genes are more divergent than the Jk genes and vary in length, encoding either 15 or 17 amino acid residues. Because JH regions comprise part of the third hypervariable region (HV3), combinatorial VH--JH joining substantially augments VH diversity. Moreover, a VH gene can recombine with each JH gene at several positions, and either one or two germ-line JH codons can be excised. This JH truncation markedly reduces the length of HV3 and hence must alter antigen-binding specificity. We have also determined the sequence of the JH4 region in two different gamma 2a mRNAs and have found that each has suffered a point mutation (aspartate to asparagine) which would alter the charge of the antigen-binding site.
Expression of biological effector functions by immunoglobulin G molecules lacking the hinge region.
Authors: Klein M , Haeffner-Cavaillon N , Isenman D E , Rivat C , Navia M A , Davies D R , Dorrington K J
Published: May 2006

Several biological effector functions mediated by sites on the Fc region of human IgG1 have been studied in two variant IgG1 kappa monoclonal proteins (Dob and Lec) which contain deletions corresponding to the entire hinge region of the heavy chains. Neither Dob nor Lec protein in aggregated form was able to activate the classical complement pathway, and this was shown to be due to an inability to bind the first component of complement (C1). By rosette inhibition assays, Dob and Lec proteins were shown to have no measurable affinity for Fc receptors on human B cells or neutrophils. Dob and Lec proteins had a much reduced affinity for Fc receptors on the murine macrophage-like cell line P388D1 when compared to normal human IgG1. Furthermore, the hinge-deleted proteins were able to compete with murine IgG2b for P388D1 receptors but not with murine IgG2a. In contrast, the binding of Dob and Lec proteins to protein A from Staphylococcus aureus was entirely normal. The functional consequences of the hinge deletion were parallel to those seen when normal IgG1 was reduced and alkylated. It was concluded that the functional impotency of Dob and Lec proteins was related to the close association between the Fab and Fc regions in these molecules and the limited degree of segmental flexibility permitted in the absence of the hinge region. The data also suggest a major role for the C gamma 2 domain (C is the constant region) in mediating effector functions in normal IgG1.
Expression of mu and gamma immunoglobulin heavy chains in different cells of a cloned mouse lymphoid line.
Authors: Burrows P D , Beck G B , Wabl M R
Published: May 2006

A cloned cell line derived from mouse bone marrow and transformed by Abelson virus is shown to synthesize two different heavy chains, mu and gamma 2B, in vitro. This characteristic is stable because it persists upon subcloning. Although most of the immunoglobulin-synthesizing cells produce either mu or gamma 2B heavy chains, a few cells contain both heavy chains, suggesting immunoglobulin class switching. Karyotypes show a complement of 41 chromosomes. Two copies of chromosome 12, to which immunoglobulin heavy chain structural genes have been assigned, were found. No light chain was found in either the mu- or the gamma 2B-producing cells. However, fusion of the cell line with a myeloma that synthesizes neither heavy nor light chains caused expression of kappa light chain in the hybridoma synthesizing mu chain. No light chain could be detected in the hybridomas synthesizing gamma 2B heavy chain.
Enzymatic treatment transforms trypomastigotes of Trypanosoma cruzi into activators of alternative complement pathway and potentiates their uptake by macrophages.
Authors: Kipnis T L , David J R , Alper C A , Sher A , da Silva W D
Published: May 2006

In the absence of bound antibody, trypomastigote bloodstream forms of Trypanosoma cruzi fail to activate the alternative complement pathway. We now demonstrate that treatment with trypsin and, to a lesser extent, with sialidase converts these protozoa into activators of the pathway, as judged by their lysis in normal sera or sera genetically deficient in fourth or second component of complement (C4 or C2) and their Mg2+-dependent consumption of C3 as measured by crossed immunoelectrophoresis. In addition, after pretreatment with enzyme and incubation in C5-deficient serum, trypomastigotes were shown to possess both C3 and properdin factor B (B) on their surface as judged by immunofluorescence. Requirement for the late components C5-C9 was suggested by the failure of C5-deficient sera to lyse trypsin-treated parasites. The inability to activate the alternative complement pathway was regained by these organisms after incubation in vitro. This restoration of insusceptibility was inhibited when puromycin was included in the culture medium. Treatment of the trypomastigotes with trypsin also potentiated their uptake by mouse peritoneal macrophages without apparent interference with their capacity to differentiate and multiply inside the cell. These findings suggest that untreated trypomastigotes normally escape recognition by the alternative pathway in vivo because of the presence on their surface of trypsin- and sialidase-sensitive regulatory molecules, the expression of which is dependent on protein synthesis.
Selective removal of apolipoprotein B-containing serum lipoproteins from blood plasma.
Authors: Stoffel W , Demant T
Published: May 2006

Studies were undertaken to determine the applicability and effectiveness of immunoadsorption chromatography on anti-low density lipoprotein (LDL) columns as a plasma-cholesterol-lowering procedure. Mass production and isolation of monospecific antibodies against swine LDL (rho = 1.006-1.063 g/ml) was carried out by immunization of sheep with swine LDL and selective antibody adsorption from their antisera by chromatography on LDL-Sepharose. The isolated LDL antibodies were then covalently linked to Sepharose CL-4B. Pig plasma LDL was effectively removed in vitro by the anti-LDL-Sepharose beads. In vivo studies were performed in pigs with (i) a plasma-separator membrane permeable to solutes below Mr 2,000,000 or (ii) a blood centrifuge interposed into an arteriovenous shunt to separate the corpuscular elements of blood from the plasma. In either case, the plasma was passed through the anti-LDL-Sepharose column and recirculated into the venous part of the shunt. Plasma cholesterol levels were reduced 70-80% by this procedure and rebounded 3-4 days after the extracorporeal immunoadsorption procedure. This continuous plasma separation-immunoadsorption procedure may have broad applicability for the elimination of any plasma component with antigenic properties.
Muscle beta-actinin and serum albumin of the chicken are indistinguishable by physicochemical and immunological criteria.
Authors: Heizman C W , Müller G , Jenny E , Wilson K J , Landon F , Olomucki A
Published: May 2006

Chicken muscle beta-actinin is considered to be one of the "true" myofibrillar components due to its specific binding to isolated myofibrils. Surprisingly, the direct comparison of this muscle protein with serum albumin, both isolated from chicken, showed that they behaved identically under several electrophoretic conditions. Furthermore, immunoreplica gels and double-immunodiffusion tests with antibodies prepared against beta-actinin established the serological identity of both proteins. No significant differences were found by circular dichroic spectroscopy or in amino acid composition. In addition, the amino-terminal sequences of both proteins were identical (H2N-Asp-Ala-Glu-His-Lys-Ser-Glu-Ile-Ala-His-Arg-Tyr-Asn-Asp-Leu-). Combined, these results strongly indicate that muscle beta-actinin and serum albumin are similar, if not identical.
Biosynthesis of vitamin B12: mode of incorporation of factor III into cobyrinic acid.
Authors: Nussbaumer C , Imfeld M , Wörner G , Müller G , Arigoni D
Published: May 2006

Extensive chemical degradation of cobyrinic acid biosynthesized from a sample of factor III radiochemically labeled in its three methyl groups demonstrates that label is retained exclusively in the two methyl groups at positions C-2 and C-7 and confirms that the C-20 methyl group of the precursor is lost during formation of the corrin ring system.
Application of differential scanning microcalorimetry to the study of cellular processes: heat production and glucose oxidation of murine macrophages.
Authors: Loike J D , Silverstein S C , Sturtevant J M
Published: May 2006

Differential scanning microcalorimetry provides a noninvasive method for studying heat evolution in living cells. We used this technique to measure the heat evolved by thioglycollate broth-elicited mouse macrophages, and the effects of NaF, KCN, cycloheximide, and cytochalasins B and D on this parameter. The total heat evolved in the interval 10--37 degrees C scanned at 1 degree C min-1 ranged from 300 to 2500 X 10(-12) cal (1 Cal = 4.184 J) per cell, depending on cell density, glucose concentration, and the presence or absence of various drugs.
Methotrexate-resistant Chinese hamster ovary cells have amplified a 135-kilobase-pair region that includes the dihydrofolate reductase gene.
Authors: Milbrandt J D , Heintz N H , White W C , Rothman S M , Hamlin J L
Published: May 2006

For the eventual purpose of isolating and studying a single animal cell replicon, we have developed a methotrexate-resistant Chinese hamster ovary cell line that has amplified an early-replicating DNA sequence approximately 500 times; this sequence includes the gene coding for dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). DHFR composes 30% of the cytoplasmic protein in this cell line, and DHFR mRNA represents 25% of the message translatable in vitro. After digestion of genomic DNA from resistant cells with restriction enzymes, a unique set of highly repetitive restriction fragments can be visualized on agarose gels by ethidium bromide staining. These bands are not present in digests of parental DNA. We estimate the total length of the unit repeated sequence to be 135 +/- 15 kilobase pairs. Regardless of the restriction enzyme utilized, a subset of these repetitive fragments hybridizes to radioactive DHFR cDNA. The homogeneously staining regions on mitotic chromosomes in which these amplified sequences are located are shown to be early-replicating, as are the highly repeated restriction fragments themselves. These data suggest that an early replicon can be isolated from this region, and that this entire, normally unique, genomic segment can be cloned and mapped with respect to origins of DNA synthesis and promoters for transcription, as well as other genetic features of interest.
Posttranslational processing of methyl-accepting chemotaxis proteins in
Escherichia coli
Authors: Sherris David , Parkinson John S.
Published: May 2006

Methyl-accepting chemotaxis proteins (MCPs) of
Escherichia coli
undergo changes in methylation state in response to chemical stimuli. The addition of methyl groups to MCP is dependent on
cheR
function; their removal is dependent on
cheB
function. This MCP methylation system is instrumental in establishing the unstimulated swimming pattern of
E. coli
and in enabling the cell to carry out sensory adaptation after a chemotactic response. We employed electrophoresis in sodium dodecyl sulfate-containing polyacrylamide gels to analyze MCP molecules synthesized in
cheR
deletion mutants lacking MCP-specific methyltransferase activity. MCP made under these conditions proved to be completely devoid of methyl groups. In the absence of
cheB
function as well, this unmethylated MCP is made in a form, designated 2
*
, that exhibits several properties characteristic of methylated MCP. In the presence of
cheB
function, MCP 2
*
is processed to a form, designated 1
*
, that no longer resembles methylated MCP. The rate of this conversion process is modulated by chemotactic stimuli. Both MCP 1
*
and MCP 2
*
are capable of initiating changes in flagellar rotation in response to stimuli, and, in the presence of
cheR
function, both forms can accept methyl groups. We suggest that MCP 2
*
is a normal intermediate in MCP synthesis in which one or more of the methyl-accepting glutamic acid residues carry a methyl-ester-like modification, which, like glutamic acid methyl esters, can be removed by
cheB
function. This
cheB
-dependent processing event does not appear to be reversible, but nevertheless it may play an important role in modulating the signaling behavior of newly synthesized MCP molecules.
Synthesis of stable unspliced mRNA from an intronless simian virus 40--rat preproinsulin gene recombinant.
Authors: Gruss P , Efstratiadis A , Karathanasis S , König M , Khoury G
Published: May 2006

We constructed a recombinant DNA molecule carrying the coding region and the 3' flanking sequences of the rat preproinsulin I gene, replacing the late region of a simian virus 40 (SV40) viral vector. The sequences making up the single intron of the preproinsulin I gene, located within the nontranslated leader region, as well as all of the 5' flanking sequences, have been deleted. The sequences remaining within the late transcriptional unit of SV40 do not retain a functional intron. This "intronless" mutant nevertheless directs the synthesis of substantial quantities of unspliced stable hybrid transcripts that are translated into polypeptides with insulin immunoreactivity. Two novel 5' termini, not used in wild-type SV40 transcripts, have been detected in the majority of the hybrid RNA molecules. These are located at analogous positions in both of the late SV40 72-base-pair repeats.
Rate of lateral diffusion of intramembrane particles: measurement by electrophoretic displacement and rerandomization.
Authors: Sowers A E , Hackenbrock C R
Published: May 2006

A method combining electrophoresis and freeze-fracture electron microscopy is described; the method was used to determine the lateral diffusion coefficient of intramembrane particles (integral proteins) in the mitochondrial inner membrane. An electric current was passed through microsuspensions of purified, spherical inner membranes at pH 7.4, which caused an electrophoretic migration of intramembrane particles in the membrane plane into a single, crowded patch facing the positive electrode. The membrane microsuspensions were quick-frozen at specified times after the packed particles were released from the electrophoretic force and while the particles were diffusing back to a random distribution. Observed concentration gradients of intramembrane particles during this time were quantitatively compared with and found to follow a mathematical model for Fickian diffusion of particles on a spherical membrane. The results determine the kinetics of free diffusion of integral proteins at the resolution of individual proteins. The diffusion coefficient of the integral proteins in the mitochondrial inner membrane was determined to be 8.3 X 10(-10) cm2/sec at 20 degrees C, from which a root-mean-square displacement of 57 nm in 10 msec is predicted.
Metabolism of benzo[a]pyrene by human mammary epithelial cells: toxicity and DNA adduct formation.
Authors: Stampfer M R , Bartholomew J C , Smith H S , Bartley J C
Published: May 2006

Pure cultures of human breast epithelial cells and of fibroblastic cells in early passage provided the opportunity to ask whether either cell type had the capability for metabolizing chemical carcinogens and, if so, was the fate of the metabolic products compatible with chemical carcinogens being a factor in the initiation of breast cancer in women. For this purpose, cells were exposed to benzo[a]pyrene (BaP), and (i) the influence on growth potential and (ii) the extent, type, and persistence of adducts between the metabolites of BaP and DNA were measured. Compared with fibroblasts, inhibition of growth by epithelial cells was 50-100 times more sensitive to BaP. Because of this differential sensitivity, epithelial cells were exposed to 0.4 microM BaP and fibroblasts were exposed to 4.0 microM BaP in the studies of DNA adduct formation. Separation by high-pressure liquid chromatography of adducts between (+/-)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP diol epoxide) and nucleosides from purified DNA revealed that epithelial cells contained modified DNA within 6 hr after adding BaP. Adducts between the 7R anti stereoisomer of BaP diol epoxide and deoxyguanosine predominated at all times. syn BaP diol epoxide adducts with deoxyguanosine and what appeared to be BaP diol epoxide adducts with deoxycytidine were consistently present but at much lower frequency. All three types of BaP diol epoxide--DNA adducts persisted in epithelial cells for 72 hr in BaP-free medium. No adducts were detected in fibroblastic cultures until 96 hr after first exposure to BaP. At this time, the type and extent of BaP diol epoxide--DNA adduct formation was similar to that in epithelial cells exposed to one-tenth the dose of BaP. The type, extent, rate of formation, and persistence of the adducts in human breast epithelial cells was similar to that in cells transformable by exposure to BaP, an indication that they may be targets for chemically induced carcinogenesis.
Lectins activate lymphocyte pyruvate dehydrogenase by a mechanism sensitive to protease inhibitors.
Authors: Beachy J C , Goldman D , Czech M P
Published: May 2006

The mitogenic lectins concanavalin A and phytohemagglutinin were found to stimulate pyruvate oxidation in rat mesenteric lymphocytes. Marked cell agglutination accompanied this response. Wheat germ agglutinin, a nonmitogenic lectin, also aggregated lymphocytes but did not cause alteration of pyruvate oxidation. Cell lysates from lectin-treated cells retained their ability to oxidize pyruvate at an elevated rate, indicating that the observed stimulation of pyruvate oxidation was not due to increased transport of labeled pyruvate into the cells. Pyruvate oxidation activity in such lysates was readily sedimented in a mitochondria-enriched cellular fraction, indicating that it reflects mitochondrial pyruvate dehydrogenase. Stimulation of this activity by lectins in intact lymphocytes was inhibited when the cells were incubated under conditions expected to inhibit trypsin-like proteases. Thus, esters of arginine, but not of alanine or tyrosine, blocked stimulation of pyruvate dehydrogenase by the lectins. The data indicate that pyruvate dehydrogenase is activated in lymphocytes treated with mitogenic lectins by a mechanism involving one or more proteolytic reactions. The similarity between the results presented here and those recently reported for insulin action on its target cells [Seals, J. R. & Czech, M. P. (1980) J. Biol. Chem. 255, 6529-6531] suggests that these systems may have similar modes of transmembrane signalling.
Proglumide and benzotript: members of a different class of cholecystokinin receptor antagonists.
Authors: Hahne W F , Jensen R T , Lemp G F , Gardner J D
Published: May 2006

In dispersed acini from guinea pig pancreas, proglumide (DL-4-benzamido-N, N-dipropylglutaramic acid) and benzotript (N-p-chlorobenzoyl-L-tryptophan) caused a rightward shift in the dose--response curve for cholecystokinin-stimulated amylase secretion but did not alter the maximal increase in amylase secretion caused by cholecystokinin. At relatively low concentrations, proglumide did not alter the stimulation of enzyme secretion caused by secretagogues whose effects are mediated by adenosine 3'5'-monophosphate (e.g., vasoactive intestinal peptide or secretin) and did not alter the stimulation of enzyme secretion caused by secretagogues that have a mode of action similar to that of cholecystokinin but act through different receptors (e.g., bombesin, physalaemin, eledoisin, and ionophore A23187). There was a close correlation between the ability of proglumide or benzotript to inhibit binding of 125I-labeled cholecystokinin to its receptors on pancreatic acini and the abilities of these compounds to inhibit the action of cholecystokinin on enzyme secretion and on calcium outflux. These results indicate that proglumide and benzotript are members of a different class of cholecystokinin receptor antagonists.
Studies of the locus for androgen receptor: localization on the human X chromosome and evidence for homology with the Tfm locus in the mouse.
Authors: Migeon B R , Brown T R , Axelman J , Migeon C J
Published: May 2006

We have established a cell line from mouse kidney cells expressing the tfm mutation and showed that these cells lack androgen binding activity. A subclone of these simian virus 40 (SV40)-transformed cells (6TGR-SV-tfm) selected in 6-thioguanine and lacking hypoxanthine phosphoribosyltransferase was used to produce a series of mouse--human hybrids containing the normal human X chromosome or various X autosome-translocation chromosomes (expressing only segments of the human X chromosome). When the androgen receptor locus (AR) was present in the hybrid, the number of receptor sites and kinetics of binding were similar to that in the human parental cells. Analysis of hybrids with partial human X chromosomes by using assays for X chromosome-linked enzymes and for the androgen receptor protein indicate that the AR locus on the human X chromosome is near the centromere between Xq13 and Xp11 and is proximal to the locus for phosphoglycerate kinase. Hybrids derived from 6TGR-SV-tfm mouse cells and human labial fibroblasts from an XY individual with the ar- form of androgen insensitivity have no binding activity. The lack of complementation indicates that the X chromosome-linked mutations in mouse and man affect homologous loci and supports the evolutionary conservation of X chromosomal loci in mammals; however, the position of the locus on the human X chromosome indicates that intrachromosomal rearrangement has occurred.
Potential for selection among nearly neutral allozymes of 6-phosphogluconate dehydrogenase in Escherichia coli.
Authors: Hartl D L , Dykhuizen D E
Published: May 2006

Six gnd alleles coding for naturally occurring allozymes of 6-phosphogluconate dehydrogenase [6-phospho-D-gluconate:NAD(P)+ 2-oxidoreductase, EC 1.1.1.43] have been transferred by transduction into the genetic background of Escherichia coli K-12 and examined for their selective effects in chemostats in which gluconate was limiting. Four of the alleles are evidently neutral or nearly neutral, inasmuch as their selective effects, if any, fall below the limit of resolution of the procedure--0.5%/hr or about 1% per generation. One allele is detrimental in limiting gluconate but not in limiting glucose or fructose. Another allele has a detrimental, density-dependent, epistatic interaction with tonA. We suggest that all six alleles are neutral or nearly neutral in natural populations but that they are not functionally equivalent; their functional differences are potentially important because they can become expressed as differences in fitness under the appropriate conditions of environment or genetic background. Under these conditions, otherwise neutral alleles can become subject to selection.
Prevention of neonatal hyperbilirubinemia by tin protoporphyrin IX, a potent competitive inhibitor of heme oxidation.
Authors: Drummond G S , Kappas A
Published: May 2006

The effects of various metalloporphyrins on hepatic heme oxygenase (EC 1.14.99.3) activity were examined in order to identify compounds that could inhibit heme degradation to bile pigment and might therefore be utilized to suppress the development of hyperbilirubinemia in the newborn. Among nine metal-protoporphyrin IX chelates (i.e., metal-hemes) studied, Sn-heme, Mn-heme, and Zn-heme substantially diminished heme oxygenase activity in vivo in the rat. These metalloporphyrins act as competitive inhibitory substrates in the heme oxygenase reaction but are not themselves oxidatively degraded. Sn-heme was the most potent enzyme inhibitor (Ki = 0.011 microM) in liver, spleen, kidney, and skin. Sn-heme administered to newborn animals within the first 72 hr after birth blocked the postnatal increase in heme oxygenase activity that occurs in various tissues. Its effect on the enzyme levels was prompt and protracted. Sn-heme administration also entirely prevented the development of hyperbilirubinemia that normally occurs postnatally. The effect of the metalloporphyrin in lowering the increased concentrations of serum bilirubin in neonates was prompt (within 1 day) and persisted throughout the 42 days after birth. No deleterious effects of Sn-heme treatment of the newborn were observed. This demonstrates that a synthetic metalloporphyrin that is a potent competitive inhibitor of heme oxidation can, when administered to the newborn, also prevent the hyperbilirubinemia that normally develops postnatally. The potential clinical implications of these findings are evident, and it is suggested that the pharmacological properties of Sn-heme and related synthetic metalloporphyrins merit further study.
Linear information processing in the retina: a study of horizontal cell responses.
Authors: Tranchina D , Gordon J , Shapley R , Toyoda J
Published: May 2006

A basic question about visual perception is whether the retina produces a faithful or a distorted neural representation of the visual image. It is now well known that in some retinal pathways there are significant nonlinear transductions which distort the neural image. The next natural question is, What are the locations of the nonlinear stages within the retinal network? We report here on an investigation of linearity and nonlinearity of responses of horizontal cells in the turtle retina as an assay of the degree of nonlinearity in the outer plexiform layer of the retina. The visual stimuli were sinusoidal gratings; these patterns were modulated by contrast reversal with a sinusoidal time course. The conclusion from our experiments is that the turtle's horizontal cell responses show evidence only of linear spatial summation even at moderately high contrasts on moderately high background levels. Our work thus indicates that there is no significant distortion of the visual image by the photoreceptors or by the neural summation of photoreceptor signals by horizontal cells under normal physiological conditions. These results are consistent with the view that the major nonlinearities of the retina are proximal to the outer plexiform layer.
Chemistry of acetylene on platinum (111) and (100) surfaces
Authors: Muetterties E. L. , Tasi M.-C. , Kelemen S. R.
Published: May 2006

An ultra-high vacuum experimental study of acetylene chemisorption on Pt(111) and Pt(100) and of the reaction of hydrogen with the acetylene adsorbate has established distinguishing features of carbon-hydrogen bond breaking and making processes as a function of pressure, temperature, and surface crystallography. The rates for both processes are substantially higher on the Pt(100) surface. Net acetylene-hydrogen processes, in the temperature range of 20°C to ≈130°C, are distinctly different on the two surfaces: on Pt(100) the net reaction is hydrogen exchange (
1
H-
2
H exchange) and on Pt(111) the only detectable reaction is hydrogenation. Stereochemical differences in the acetylene adsorbate structure are considered to be a contributing factor to the differences in acetylene chemistry on these two surfaces.
Scrapie agent contains a hydrophobic protein.
Authors: Prusiner S B , McKinley M P , Groth D F , Bowman K A , Mock N I , Cochran S P , Masiarz F R
Published: May 2006

The scrapie agent causes a degenerative nervous system disorder of sheep and goats. Considerable evidence indicates that the scrapie agent contains a protein that is necessary for infectivity [Prusiner, S. B., Groth, D. F., Cochran, S. P., Masiarz, F. R., McKinley, M. P. & Martinez, H. M. (1980) Biochemistry 19, 4883-4891], but direct demonstration of a protein moiety has been hampered by lack of sufficiently purified preparations. Employing preparations of the scrapie agent enriched 100- to 1000-fold with respect to protein, we found that digestion by proteinase K destroyed more than 99.9% of the infectivity. Diethylpyrocarbonate, which chemically modifies amino acid residues in proteins with high efficiency, also inactivated the scrapie agent in these purified preparations. Reductions of infectivity by proteinase K and diethylpyrocarbonate were not observed with less purified preparations. The agent bound to phenyl-Sepharose could not be eluted with 8.5 M ethylene glycol; however, a combination of ethylene glycol and detergents did release the agent. These observations provide good evidence for a protein and for hydrophobic domains within the scrapie agent. Whether the protein required for infectivity is the same protein responsible for the hydrophobic properties of the scrapie agent remains to be established.
K
+
/Na
+
-triggered bioluminescence in the oceanic squid
Symplectoteuthis oualaniensis
Authors: Tsuji Frederick I. , Leisman Gary B.
Published: May 2006

A distinctive type of luminescent system present in the large dorsal luminous organ of the oceanic squid
Symplectoteuthis oualaniensis
is described. The organ produces an intense blue flash of light followed by a rapid decay in light intensity. Luminescence originates from numerous oval granules present in the luminous organ. The essential light-emitting components are membrane bound. Intact granules or washed homogenates of the granules are triggered to emit light by monovalent cations such as, in decreasing order of effectiveness, potassium, rubidium, sodium, cesium, ammonium, and lithium. Calcium, magnesium, and strontium ions do not trigger light emission. Analysis of the kinetics of the decay of light intensity suggests that two light-emitting components are involved, one decaying faster than the other. The light-emitting reaction has an absolute requirement for molecular oxygen. The optimum KCl or NaCl concentration is ≈0.6 M and the optimum pH is ≈7.8. A free sulfhydryl group is essential for activity.
Evidence that a high molecular weight replicative DNA polymerase is conserved during evolution.
Authors: Hübscher U , Spanos A , Albert W , Grummt F , Banks G R
Published: May 2006

Using a technique developed recently to detect DNA polymerase activity in situ after NaDodSO4 gel electrophoresis (Spanos, A., Sedgwick, S. G., Yarranton, g. T., Hübscher, U. & Banks, G. R. (1981) Nucleic Acids Res. 9, 1825-1839), we present evidence that a high Mr (greater than or equal to 125,000) polypeptide is responsible for chromosomal DNA replication in prokaryotes, lower eukaryotes and high eukaryotes. Not only extracts from Escherichia coli, Ustilago maydis, Drosophila melanogaster, rat neurones, calf thymus, human fibroblast, and HeLa cells possess such high Mr activities, but also highly purified E. coli DNA polymerase III core enzyme, U. maydis DNA polymerase, and D. melanogaster embryo and calf thymus DNA alpha polymerases. The evidence that these activities are responsible for chromosomal DNA replication is genetical (E. coli, U. maydis, and D. melanogaster); also, the high Mr activity disappears from rat neurones during differentiation from an actively dividing precursor cell to a postmitotically mature neurone. Furthermore, when limited proteolysis is allowed to occur, a defined and remarkably similar pattern of intermediate Mr activities is generated in lower eukaryotic and high eukaryotic extracts and, to some extent, in prokaryotic extracts. In higher eukaryotic extracts, a low Mr activity of approximately 35,000 is also generated. Protease inhibitors can retard formation of these catalytically active proteolytic fragments. We propose that the replicative DNA polymerase complex of both prokaryotes and eukaryotes contains a high Mr polypeptide responsible for chain elongation which might be conserved during evolution and which is extremely sensitive to proteolytic cleavage.
Effect of tunicamycin on appearance of carbohydrate variants of plasminogen in rat and rabbit plasma.
Authors: Powell J R , Bretthauer R K , Castellino F J
Published: May 2006

The in vivo incorporation of [4,5-3H]leucine and [2-3H]mannose into the lysine-Sepharose affinity chromatography-resolvable carbohydrate variants of rat and rabbit plasminogen has been studied in the absence and presence of tunicamycin (TM). When rabbits and rats were exposed to 0.5 mg of TM per kg of body weight for 30 min and 2 hr, respectively, followed by a single pulse of [4,5-3H]leucine for 2 hr, no radiolabel was found in rat or rabbit variant 1 plasminogen, whereas [4,5-3H]leucine was incorporated into variant 2 plasminogen. In similar experiments utilizing [2-3H]mannose in place of [4,5-3H]leucine, very little [2-3H]mannose was incorporated into either plasminogen variant in the presence of TM. The trichloroacetic acid-precipitable protein of rabbit plasma incorporated virtually identical quantities of [4,5-3H]leucine in the absence or presence of TM, under the above dosage regimens, while, at the same time, a large decrease in the incorporation of [2-3H]mannose was observed in the plasma proteins of rabbits treated with TM. This indicates that many of the plasma proteins may be secreted into plasma divested of asparagine-linked carbohydrate side chains. Further, we conclude that the functional difference (antifibrinolytic amino acid binding capacity) between the two plasma variants of plasminogen more than likely results from additional glycosylation of asparagine in variant 1 plasminogen that normally does not occur in variant 2 plasminogen.
Cytoplasmic utilization of liposome-encapsulated myosin heavy chain messenger ribonucleoprotein particles. During muscle cell differentiation.
Authors: Havaranis A S , Heywood S M
Published: May 2006

Myosin heavy chain messenger ribonucleoprotein particles (MHC mRNPs) have been isolated. Characterization of the RNA components revealed an mRNA of approximately the same size as tobacco mosaic virus RNA and three low molecular weight components. The protein consists of 9-10 major bands ranging in molecular weight between 22,000 and 130,000. The messenger contained in these mRNPs was found to direct the synthesis of both fast-muscle and slow-muscle MHC in a cell-free system. When MHC [3H]mRNPs were encapsulated into liposomes and subsequently delivered to myoblasts and myotubes, the mRNPs were taken up by the cells at both stages of differentiation. However, the MHC [3H]mRNPs taken up by the myoblasts remained as free cytoplasmic particles (80-120S), whereas in myotubes the incorporated mRNP RA was associated with polysomes. The results indicate that MHC mRNPs contain a repressor molecule(s) and that myotubes possess a mechanism for activating these mRNPs that is absent from myoblasts.
Insulin-induced receptor loss in cultured human lymphocytes is due to accelerated receptor degradation.
Authors: Kasuga M , Kahn C R , Hedo J A , Van Obberghen E , Yamada K M
Published: May 2006

We have measured the turnover rate of the polypeptide subunits of the insulin receptor in cultured human lymphocytes (IM-9 line) and have investigated the mechanism of insulin-induced receptor loss. To estimate the rate of receptor degradation, lymphocytes were either pulse-labeled with [35S]methionine or surface labeled with Na125I and lactoperoxidase. The insulin receptor was isolated by immunoprecipitation with anti-receptor antibody, and the rate of loss of radioactivity from each receptor subunit was determined after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Two major (Mr 135,000 and 95,000) and one minor (Mr 210,000) subunits were found. By both labeling methods, the half-lives of the major insulin receptor subunits were 9--12 hr in normal media. When the cells were cultured in media containing 1 microM insulin the turnover was accelerated 2.5- to 3.5-fold (half-life approximately 3 hr). The increase in degradation rate was dependent on the insulin concentration and correlated well with the ability to "down-regulate" the receptor. Guinea pig insulin was about 2% as active as porcine insulin in accelerating degradation, and human growth hormone was without effect. The acceleration of receptor degradation induced by insulin was partially blocked by 100 microM cycloheximide. The rate of biosynthesis of the insulin receptor did not appear to be altered in the presence of 1 microM insulin after correction for the change in degradation rate. In conclusion, these data demonstrate that insulin-induced receptor loss in cultured lymphocytes is due to accelerated receptor degradation.
Carbohydrate-specified endocytosis: localization of ligand in the lysosomal compartment.
Authors: Haimes H B , Stockert R J , Morell A G , Novikoff A B
Published: May 2006

Carbohydrate-directed endocytosis is mediated by a receptor, the hepatic binding protein; it is responsible for the clearance of galactose-terminated glycoproteins from the circulation. This process was investigated by using lactosaminated ferritin which is recognized by this receptor. Ferritin was seen in elements of an extensive "lysosomal compartment" that includes secretory vacuoles, coated vesicles, and GERL. The compartment marked in hepatocytes by the distribution of ligand is similar to that previously described in Kupffer cells (marked by acid phosphatase reaction product and horseradish peroxidase reaction product).
Stochastic expression of fetal hemoglobin in adult erythroid cells.
Authors: Stamatoyannopoulos G , Kurnit D M , Papayannopoulou T
Published: May 2006

The expression of fetal hemoglobin, Hb F, in the adult cells is cellularly restricted both in vivo and in culture. Because, in cultures of erythroid progenitors, subclones that express or fail to express Hb F are derived from the same erythroid stem cell, a mechanism must exist whereby Hb F expression segregates in the progeny of erythroid progenitors during their differentiation. We present mathematical analyses of experimental data which suggest that expression of Hb F in human adult erythroid cells occurs on a stochastic basis. We quantified Hb F expression among the subclones of erythroid bursts (clones) in vitro by labeling subclones with fluorescent anti-Hb F antibodies. The observed data were compared with predictions from a stochastic model with the assumption that the expression of Hb F in a subclone occurs with a probability P equal to the frequency of Hb F-expressing subclones in an experiment. There was good fit between the observed and predicted data with respect to: (i) the relative frequencies of monomorphic F+, monomorphic F-, and bimorphic F+/F- bursts, respectively; (ii) the size distributions among F+, F- and F+/F- bursts; and (iii) the proportions of subclones expressing Hb F within bimorphic F+/F- bursts. Given the hypothesis that erythroid progenitors which have an active Hb F program are less differentiated than cells which do not proceed to express Hb F, the stochastic event indicated by our analyses may be the probability that adult erythroid progenitor cells undergo terminal differentiation at an earlier stage than usual.