bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective hepatic uptake of human β-hexosaminidase A by a specific glycoprotein recognition system on sinusoidal cells
Authors: Steer Clifford J. , Kusiak John W. , Brady Roscoe O. , Jones E. Anthony
Published: May 2006

Intravenously administered
125
I-labeled human β-hexosaminidase A (β-
N
-acetylglucosaminidase; 2-acetamido-2-deoxy-β-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) was rapidly cleared from the circulation of rats and accumulated in the liver. When hepatic cells were subsequently isolated, the label was recovered from both sinusoidal cells and, to a lesser extent, hepatocytes. Clearance was inhibited by the simultaneous infusion of mannan but not by a galactose-terminated glycoprotein. Studies
in vitro
, in which
125
I-β-hexosaminidase was incubated with isolated hepatic cells, detected no uptake of the labeled ligand by hepatocytes. In contrast, uptake by sinusoidal cells was shown to be temperature dependent and approached saturability. Prior treatment of sinusoidal cells with Pronase resulted in markedly decreased uptake of
125
I-β-hexosaminidase by these cells. Mannan and partially deglycosylated glycoproteins bearing terminal nonreducing
N
-acetylglucosamine or mannose residues were shown to be potent inhibitors of the cellular uptake of
125
I-β-hexosaminidase; native orosomucoid and desialylated (galactoseterminated) orosomucoid were not inhibitory. Of six simple sugars tested, including
N
-acetylglucosamine, only mannose was an effective inhibitor of the cellular uptake of
125
I-β-hexosaminidase. The kinetics of uptake of β-hexosaminidase and mannose-terminated orosomucoid by sinusoidal cells were shown to be similar. These findings suggest that the hepatic uptake of the lysosomal glycosidase β-hexosaminidase A is mediated by a receptor on sinusoidal cells which recognizes and binds mannose-terminated glycoproteins.
Immunocytochemical localization of alpha-actinin in intestinal epithelial cells.
Authors: Geiger B , Tokuyasu K T , Singer S J
Published: May 2006

alpha-Actinin was localized in chicken intestinal epithelial cells by immunofluorescence and immunoferritin labeling of thin frozen sections. Most of the label of the brush border was confined to the terminal web area. The label there was concentrated mainly along the "roots" of the microvilli core microfilaments and in the vicinity of the zonula adherens. In the latter structure, the narrow electron-dense zones adjacent to the cell membranes, however, were not significantly labeled. This suggests that alpha-actinin does not mediate directly the association of the transverse terminal web microfilaments to the membrane at the zonula adherens. Sparse ferritin labeling was found near the tight junction, whereas the staining associated with the spot desmosome was negligible. The microvilli were not significantly labeled by either immunofluorescence or immunoferritin staining unless the sections were previously treated with detergent. Moreover, alpha-actinin (or a structurally related protein) was not detected in preparations of purified microvillar vesicles, suggesting the possibility that the alpha-actinin staining in the microvilli may be an artificial due to its translocation by the detergent from the terminal web onto the microvilli. The possible roles of alpha-actinin in the organization and function of the brush border are discussed.
Two processing steps in maturation of vitellogenin polypeptides in Drosophila melanogaster.
Authors: Warren T G , Brennan M D , Mahowald A P
Published: May 2006

Synthesis of the three vitellogenin polypeptides (molecular weights of 44,000, 45,000, and 46,000) of Drosophila melanogaster has been analyzed in vivo and in a cell-free system. After labeling periods in vivo, the three vitellogenin polypeptides were made as the principal synthetic products of the female fat body. During a short (0.5 min) labeling period, they were identified as discrete species on two-dimensional gels. Two of the polypeptides have molecular weights of 45,000 and a third has a molecular weight of 44,000. After longer labeling periods (5-45 min) the three mature vitellogenins appeared. Both immunoprecipitation and peptide mapping confirmed that the species labeled at 0.5 min are immature forms of the vitellogenin polypeptides. In vitro translation of poly(A)-RNA from female fat body indicated another processing step in vitellogenin synthesis. Three polypeptides were obtained that were identified as precursors of the vitellogenins on the basis of immunoprecipitation and peptide mapping. Two of the translation products have a molecular weight of 46,000 and the third has a molecular weight of 45,000. Because the vitellogenins are secreted proteins, we interpret the higher molecular weight of the in vitro translation products as being due to signal peptides.
Virus-specified protease in poliovirus-infected HeLa cells.
Authors: Korant B , Chow N , Lively M , Powers J
Published: May 2006

Previous studies have shown that primary cleavages in nascent picornavirus precursors are accomplished by cellular proteases. This study has characterized the enzyme in infected cells that produces the capsid polypeptides by secondary cleavages of viral precursors. The kinetics of the production of protease activity correlate with the time course of virus protein synthesis, and the new enzyme has characteristic pH and temperature optima. Guanidine and cycloheximide, which are inhibitors of virus RNA and protein synthesis, prevent production of the protease. As determined by introduction of amino acid analogs into the protease or inhibition by a leucyl chloromethyl ketone, the enzyme is synthesized at a time of infection when host cell proteins are not produced, and the enzyme copurified with a 40,000-dalton virus polypeptide present in the cytoplasm of infected cells. Wild-type levels of protease activity are produced by viral mutants that are defective in coat protein synthesis. The conclusion is that a non-structural poliovirus gene product participates in protein cleavages that produce the viral coat proteins.
Recombination between the defective component of an acute leukemia virus and Rous associated virus O, an endogenous virus of chickens.
Authors: Tsichlis P N , Coffin J M
Published: May 2006

The ability of the defective acute leukemia virus of chickens, MC-29, to participate in recombination was investigated by testing the ability of the MC-29 genome to donate sequences to its helper virus. The endogenous virus Rous associated virus O (RAV-O) was used as a helper for MC-29, and its genome was compared by fingerprinting to that of the original RAV-O. In three separate isolates, it was found that the RAV-O used as helper for MC-29 had acquired new sequences near the 3' and 5' ends of its genome. The new 3' proximal sequences resembled the C region found in exogenous but not endogenous avian oncoviruses, and it probably imparted a higher growth rate to the recombinant as compared to RAV-O. One isolate also showed recombination within the env gene. Because we could exclude the possibility that the recombination was with host cell information or with the original helper of MC-29, we conclude that the acquired sequences were derived from the MC-29 genome, and therefore this replication defective virus is not defective in recombination.
Time-resolved resonance Raman spectroscopy of intermediates of bacteriorhodopsin: The
bK
590
intermediate
Authors: Terner James , Hsieh Chung-Lu , Burns A. R. , El-Sayed M. A.
Published: May 2006

We have combined microbeam and flow techniques with computer subtraction methods to obtain the resonance Raman spectrum of the short lived batho-intermediate (
bK
590
) of bacteriorhodopsin. Comparison of the spectra obtained in
1
H
2
O and
2
H
2
O, as well as the fact that the
bK
590
intermediate shows large optical red shifts, suggests that the Schiff base linkage of this intermediate is protonated. The fingerprint region of the spectrum of
bK
590
, sensitive to the isomeric configuration of the retinal chromophore, does not resemble the corresponding region of the parent
bR
570
form. The resonance Raman spectrum of
bK
590
as well as the spectra of all of the other main intermediates in the photoreaction cycle of bacteriorhodopsin are discussed and compared with resonance Raman spectra of published model compounds.
Cellular information in the genome of recovered avian sarcoma virus directs the synthesis of transforming protein.
Authors: Karess R E , Hayward W S , Hanafusa H
Published: May 2006

Recovered avian sarcoma viruses, whose sarcomagenic information is largely derived from cellular sequences [Wang, L.-H., Halpern, C.C., Nadel, M. & Hanafusa, H. (1978) Proc. Natl. Acad. Sci. USA 75, 5812-5816], produce the transforming protein p60src in infected cells, in amounts comparable to the amount found in cells transformed by standard strains of avian sarcoma virus. Though displaying some virus-specific differences in electrophoretic mobility, p60srcs from these viruses are similar to those of other avian sarcoma virus strains by the criteria of (i) antigenicity, (ii) partial proteolysis mapping, and (iii) association with protein kinase activity. We also find that p60sarc, a protein present in normal cells at a low level, is associated with a protein kinase activity, and thus it too is similar by the above criteria to p60src of avian sarcoma virus. Possible causes for the pathogenicity of p60src are discussed in light of these similarities.
Cyclic AMP as a modulator of polarity in polycistronic transcriptional units.
Authors: Ullmann A , Joseph E , Danchin A
Published: May 2006

The degree of natural polarity in the lactose and galactose operons of Escherichia coli is affected by adenosine 3',5'-cyclic monophosphate (cAMP). This effect, mediated by the cAMP receptor protein, is exerted at sites distinct from the promoter. Experiments performed with a mutant bearing a thermosensitive rho factor activity indicate that cAMP relieves polarity by interfering with transcription termination. Conflicting results in the literature concerning the role of cAMP receptor protein and cAMP in galactose operon expression can be reconciled by the findings that cAMP stimulates the expression of operator distal genes without significantly affecting the proximal genes. Therefore, it appears necessary to reevaluate the classification of the galactose operon as exhibiting cAMP-mediated catabolite repression at the level of transcription initiation.
Presence of free cyclic AMP receptor protein and regulation of its level by cyclic AMP in neuroblastoma-glioma hybrid cells.
Authors: Walter U , Costa M R , Breakefield X O , Greengard P
Published: May 2006

Neuroblastoma-glioma hybrid cells of line 108CC-5 were found to contain high levels of soluble adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase activity and high levels of two specific cAMP receptor proteins, RI and RII. Treatment of the hybrid cells with dibutyryl cAMP increased the level of RI but did not significantly affect the level either of RII or of cAMP-dependent protein kinase activity. The effect of dibutyryl cAMP could be mimicked by prostaglandin E1 and 3-isobutyl-1-methylxanthine, both of which are known to raise cAMP levels in neuroblastoma-glioma hybrid cells. Both in control as well as in dibutyryl cAMP-treated cells, RII but not RI was associated with cAMP-dependent protein kinase. Several lines of evidence suggest that RI represents the free regulatory subunit of type I cAMP-dependent protein kinase. The presence of this regulatory subunit as free cAMP receptor protein in neuroblastoma-glioma hybrid cells may be of significance with respect to the regulation of growth and differentiation in tumor cells.
Transcriptional regulation of the yeast cytochrome c gene.
Authors: Zitomer R S , Montgomery D L , Nichols D L , Hall B D
Published: May 2006

DNA from the cloned yeast iso-1-cytochrome c, cycl, gene was used in a hybridization assay to measure levels and rates of synthesis of cycl RNA. Derepressed cells synthesized cycl RNA at 6 times the rate of that of glucose-repressed cells. Upon glucose addition to a derepressed culture, the transcription of the cycl gene was repressed within 2.5 min. The half-life of hybridizable cycl RNA was determined to be 12-13.5 min under repressed and derepressed conditions and during repression. The results demonstrate that the expression of the cycl gene is subject to transcriptional regulation.
Proton nuclear magnetic resonance investigation of structural changes associated with cooperative oxygenation of human adult hemoglobin
Authors: Viggiano Giulio , Ho Chien
Published: May 2006

The structural changes associated with cooperative oxygenation of human adult hemoglobin as a function of oxygen saturation in aqueous media at neutral pH and at 25-27°C have been investigated by high-resolution proton nuclear magnetic resonance spectroscopy at 250 and 360 MHz. By monitoring the intensities of two hyperfine shifted proton resonances (at about -12 and -18 ppm from H
2
O) and two exchangeable proton resonances (at about -6.4 and -9.4 ppm from H
2
O) as a function of oxygenation, the amount of oxygen bound to the α and β chains of a hemoglobin molecule can be determined and the relationship between tertiary and quaternary structural changes under a given set of experimental conditions can be investigated. These results suggest that: (
i
) in the absence of organic phosphates, there is no preferential O
2
binding to the α or β chains; (
ii
) in the presence of organic phosphates, the α hemes have a higher affinity for O
2
as compared to the β hemes; (
iii
) the ligand-induced structural changes in the hemoglobin molecule are not concerted; and (
iv
) some cooperativity must be present within the deoxy quaternary state during the oxygenation process. The variations of the exchangeable proton resonances as a function of oxygenation strongly suggest that the breaking of one or more inter- or intrasubunit linkages of a ligated subunit can affect similar linkages in unligated subunits within a tetrameric hemoglobin molecule. Thus, the present results show that two-state allosteric models are not adequate to describe the cooperative oxygenation of hemoglobin. In addition, the present results provide direct correlation to the ligand-induced structural changes (such as in the heme pockets and subunit interfaces) observed to occur in the crystals of deoxy- and oxy-like hemoglobin molecules and in the solution state.
Identification of separate mRNAs coding for the alpha and beta subunits of thyrotropin.
Authors: Vamvakopoulos N C , Kourides I A
Published: May 2006

A 9S mRNA, purified from mouse thyrotropic pituitary tumors by sucrose density gradient centrifugation of poly(A)-enriched mRNA, directed the synthesis of only alpha and beta subunits of thyrotropin in the reticulocyte lysate translation system. Analysis of radioiodinated 9S mRNA, repurified by oligo(dT)-cellulose chromatography and sucrose gradient centrifugation, yielded two species of RNA on urea/polyacrylamide gel electrophoresis. The major RNA species contained 620 nucleotides, and the minor RNA species contained 560 nucleotides. Unlabeled 9S mRNA was further purified by urea/polyacrylamide gel electrophoresis; the mRNAs were separately eluted from slices of the gel containing material migrating with an apparent length of 620 and 560 nucleotides. Translation of these mRNAs in the reticulocyte lysate showed that the longer mRNA coded for the alpha subunit and the shorter mRNA coded for the beta subunit of mouse thyrotropin. Because more alpha than beta subunit of thyrotropin was consistently synthesized, unbalanced amounts of thyrotropin subunits appear to be synthesized by translation of unbalanced amounts of individual mRNAs. We have demonstrated that the synthesis of thyrotropin is directed by two separate mRNA molecules, each coding for a different subunit of the hormone.
Induction of cytokinin autonomy by
N,N
′-diphenylurea in tissue cultures of
Phaseolus lunatus
L
Authors: Mok Machteld C. , Kim Sang-Gu , Armstrong Donald J. , Mok David W. S.
Published: May 2006

The ability of
N,N
′-diphenylurea (Ph
2
urea) to substitute for cytokinin-active adenine derivatives in promoting callus growth of
Phaseolus lunatus
has been examined. In general, Ph
2
urea stimulated callus growth at high concentrations, although the growth of most callus tissues was irregular. Variability in the sensitivity and uniformity of the growth response to Ph
2
urea was found among different genotypes of
P. lunatus
. Most importantly, tissues cultured on Ph
2
urea-containing medium for one passage had acquired the ability to proliferate in subsequent passages in the absence of either Ph
2
urea or cytokinin-active adenine derivatives. Corresponding tissues maintained on kinetin-containing medium remained cytokinin-dependent. It appears that the effect of Ph
2
urea in promoting the growth of
P. lunatus
callus tissue resides in its ability to induce cytokinin autonomy. This result suggests that the cytokinin activity of Ph
2
urea may be due to promotion of endogenous cytokinin biosynthesis in the bioassay systems in which it is active.
Microinjection of fluorescently labeled α-actinin into living fibroblasts
Authors: Feramisco James R.
Published: May 2006

α-Actinin from chicken gizzard labeled with tetramethylrhodamine isothiocyanate has been incorporated into living fibroblast cells by microinjection. Fluorescent labeling of α-actinin was carried out such that the conjugated protein was functional
in vitro
as shown by its ability to bind to F-actin. Within 1-2 hr after injection, diffuse fluorescence was observed throughout the cytoplasm and only faint fluorescence was apparently associated with the stress fibers. During the ensuing 2-15 hr, however, most of the fluorescence was seen as periodicities along the stress fibers and as foci of the microfilament polygonal networks. This distribution of α-actinin in the living cells was strikingly similar to that found by indirect immunofluorescence localization of endogenous α-actinin in fixed samples of the same cell type. Control studies in which heat-treated (100°C, 2 min) fluorescent α-actinin or tetramethylrhodamine isothiocyanate alone was injected into the cells indicated that the stress fiber and polygonal network labeling was specific for “native” fluorescently labeled α-actinin. These results suggest that the dynamic properties of proteins and structures in cultured mammalian cells can be studied with the use of microinjection and fluorescence microscopic techniques.
Origins of metabolic diversity: Evolutionary divergence by sequence repetition
Authors: Ornston L. N. , Yeh W. K.
Published: May 2006

Recurring patterns of primary structure have been observed in enzymes that mediate sequential metabolic reactions in bacteria. The enzymes, muconolactone Δ-isomerase [(+)-4-hydroxy-4-carboxymethylisocrotonolactone Δ
2
-Δ
3
-isomerase, EC 5.3.3.4] and β-ketoadipate enol-lactone hydrolase [4-carboxymethylbut-3-enolide(1,4)enol-lactone-hydrolase, EC 3.1.1.24], have been coselected in bacterial populations because the isomerase can confer no nutritional advantage in the absence of the hydrolase. Similar amino acid sequences recur within the structure of the isomerase, and the amino-terminal amino acid sequence of the isomerase from
Pseudomonas putida
appears to be evolutionarily homologous with the corresponding sequence of a β-ketoadipate enol-lactone hydrolase from
Acinetobacter calcoaceticus
. One interpretation of the sequence repetitions is that they reflect tandem duplication mutations that took place early in the evolution of the proteins. According to this view, the mutations caused elongation of structural genes and the creation of duplicated genes as the metabolic pathways evolved. A review of the sequence data calls attention to a different hypothesis: repeated amino acid sequences were introduced in the course of the proteins' evolution by substitution of copies of DNA sequences into structural genes. Our observations are interpreted on the basis of a model proposing genetic exchange between misaligned DNA sequences. The model predicts that misalignments in one chromosomal region can influence the nature of mutations in another region. Thus, as often has been observed, the mutability of a base pair will be determined by its location in a DNA sequence. Furthermore, the intrachromosomal recombination of DNA sequences may account for complex genetic modifications that occur as new pathways evolve. The model provides an interpretation of an apparent paradox, the rapid creation of new metabolic traits by bacterial genomes that are remarkably resistant to genetic drift.
Effect of anti-HLA antisera on macrophage-T-cell interactions.
Authors: Geha R S , Milgrom H , Broff M , Alpert S , Martin S , Yunis E J
Published: May 2006

Human T lymphocytes were shown to proliferate in response to tetanus toxoid antigen only in the presence of macrophages. This response was inhibited by anti-DRw but not by anti-HLA (A and B loci) antisera added to the cultures and by pretreatment of macrophages but not of T cells with anti-DRw antisera and complement. Macrophages pulsed for 18 hr with antigen and then washed were capable of triggering T-cell proliferation. Addition of anti-DRw but not anti-HLA (A and B loci) antisera during the pulse period inhibited the macrophages' ability to trigger T-cell proliferation. The data obtained indicate that human T cells recognize and proliferate in response to antigen presented by the macrophages in association with Ia-like antigens.
Distinct
H-2
-linked
Ir
genes control both antibody and T cell responses to different determinants on the same antigen, myoglobin
Authors: Berzofsky Jay A. , Richman Lee K. , Killion Douglas J.
Published: May 2006

The murine antibody and T lymphocyte proliferative responses to sperm whale myoglobin (Mb) were found to be under control of two distinct
H-2
-linked immune response (
Ir
) genes (
Ir-Mb-1
, mapping in the
I-A
subregion, and
Ir-Mb-2
, mapping in
I-C
).

H-2
d

mice (B10.D2 and DBA/2), with both genes, were high responders to Mb and its fragments for both antibody secretion and T cell proliferation, while

H-2
b

(B10) and

H-2
k

(B10.BR) mice were low responders. Strains with only
Ir-Mb-2
[B10.A and B10.A(5R)], which were intermediate responders to Mb, made antibodies to and proliferated in response to the NH
2
-terminal fragment (1-55) but not the COOH-terminal fragment (132-153) when immunized with Mb. In contrast, mice carrying only the
Ir-Mb-1
gene (D2.GD and B10.GD) made antibodies to and proliferated in response to both fragments. However, their proliferation to fragment (1-55) was often lower than that of their congenic high responders (DBA/2 and B10.D2, respectively), possibly because they respond to only some of the determinants on this NH
2
-terminal fragment. Thus, these data demonstrate that distinct
Ir
genes, mapping in separate
I
-subregions of
H-2
, control responses to different antigenic determinants on the same protein molecule. Moreover, the gene that controls the T lymphocyte responses to a given determinant also controls production of antibodies specific for that same determinant (or a closely associated one).
Specific changes in the surface glycoprotein pattern of human promyelocytic leukemic cell line HL-60 during morphologic and functional differentiation.
Authors: Gahmberg C G , Nilsson K , Andersson L C
Published: May 2006

The human promyelocytic leukemia cell line HL-60 can be induced to undergo morphological and functional differentiation in vitro by various low molecular weight compounds. The cellular morphology changes from blastoid appearance to that of granulocytes and the cells acquire the ability to phagocytize. We here report that the surface glycoproteins specifically change during this differentiation, as shown by the neuraminidase/galactose oxidase/NaB3H4 surface-labeling technique followed by polyacrylamide slab gel electrophoresis. The most prominent change is the loss of the major glycoprotein band typical for the blast cells which has an apparent molecular weight of 160,000 and the appearance of a major surface glycoprotein band with an apparent molecular weight of 130,000. Expression of the 130,000 molecular weight band correlates with the appearance of phagocytic and chemotactic activities of the cells. It has the same molecular weight as the major surface glycoprotein of freshly isolated human blood granulocytes.
Trace polypeptides in cellular extracts and human body fluids detected by two-dimensional electrophoresis and a highly sensitive silver stain.
Authors: Merril C R , Switzer R C , Van Keuren M L
Published: May 2006

Development of a highly sensitive silver stain permits the characterization of trace cellular and body fluid proteins separated by the two-dimensional electrophoresis technique of O'Farrell. Many of the proteins detected by the silver stain in urine, spinal fluid, amniotic fluid, and cells were undetected with the widely used Coomassie blue stain. Trace polypeptides observed in Escherichia coli cell lysates with this silver stain could be detected previously only by growth in radioactive precursors followed by lengthy autoradiography. In situations that do not permit the use of radioactive labeling, as in human clinical studies, the enhanced ability to detect proteins achieved by the silver stain will facilitate metabolic studies and the screening for protein abnormalities in mutational studies and in genetic diseases.
Murine erythroleukemia cell differentiation: relationship of globin gene expression and of prolongation of G1 to inducer effects during G1/early S.
Authors: Gambari R , Marks P A , Rifkind R A
Published: May 2006

Murine erythroleukemia cells (MELC) are induced to erythroid differentiation by culture with 4 mM hexamethylenebisacetamide. The relationship between onset of accumulation of globin mRNA and cell cycle phase has been examined in MELC fractionated with respect to cell cycle (G1, early S, mid S, and late S/G2) by the technique of centrifugal elutriation. This technique provides synchronized populations of cells without use of chemicals that block cell function (e.g., DNA synthesis). It is shown that the initial onset of accumulation of globin mRNA occurs during the first G1 phase after a complete S traversed in culture with inducer. Once globin mRNA synthesis is initiated, it continues throughout the cell cycle. Studies of synchronized MELC populations exposed to hexamethylenebisacetamide starting in G1, mid S, or late S/G2, provide evidence that an effect of the inducer during G1 or early S is critical to initiation of accumulation of newly synthesized globin mRNA and to the prolongation of G1, both of which are associated with terminal erythroid differentiation.
Calculation of evolutionary trees from sequence data.
Authors: Klotz L C , Komar N , Blanken R L , Mitchell R M
Published: May 2006

Evolutionary trees are usually calculated from comparisons of protein or nucleic acid sequences from present-day organisms by use of algorithms that use only the difference matrix, where the difference matrix is constructed from the sequence differences between pairs of sequences from the organisms. The difference matrix alone cannot define uniquely the correct position of the ancestor of the present-day organisms (root of the tree). Furthermore, methods using the difference matrix alone often fail to give the correct pattern of tree branching (topology) when the different sequences evolve at different rates. Only for equal rates of evolution can the difference matrix (when used with the so-called matrix method) yield exactly the correct topology and root. In this paper we present a method for calculating evolutionary trees from sequence data that uses, along with the difference matrix, the rate of evolution of the various sequences from their common ancestor. It is proven analytically that this method uniquely determines both the correct topology and root in theory for unequal rates of sequence evolution. How one would estimate an ancestral sequence to be used in the method is discussed in particular for the 5S RNA sequences from prokaryotes and eukaryotes and for ferredoxin sequences.
Catabolite inactivation of gluconeogenic enzymes in mutants of yeast deficient in proteinase B.
Authors: Zubenko G S , Jones E W
Published: May 2006

Strains of Saccharomyces cerevisiae bearing nonsense mutations in the structural gene for proteinase B (EC 3.4.22.9) have been examined for the ability to make the transition from growth on acetate to growth on glucose and for the ability to inactivate three glucoeogenic enzymes during the transition because proteinase B has been proposed by others to be responsible for the inactivation of the three enzymes during the growth transition. The mutant strains make the growth transition normally. Catabolite inactivation of hexosediphosphatase (D-fructose-1,6-biphosphate 1-phosphohydrolase, EC 3.1.3.11), malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37), and phosphoenolpyruvate carboxykinase (ATP) [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] occurred in prb1 mutants with kinetics similar to those seen in wild-type strains. We infer that proteinase B activity is not essential for the process of catabolite inactivation.
Immunoglobulin differentiation is dictated by repeated recombination sequences within the V region prototype gene: A hypothesis
Authors: Ben-Sasson Shmuel A.
Published: May 2006

Analysis of the available DNA sequences of immunoglobulin light chain genes reveals a unique structural pattern. A stretch of about 15 nucleotides repeats five times within the variable (V) region gene, with few base changes. Identification of these homologous sequences is apparent in the embryonic V
λ
gene and might also be recognized in V
κ
genes isolated from a myeloma. Although different from each other, the V
λ
and V
κ
hyperhomologous sequences display a remarkable resemblance to different prokaryote sequences associated with recombinational events. The homologous sequences appear at all three sites where hypervariable regions of the mature peptide are encoded. In addition, they are located at the site where V/constant (C) recombination is supposed to take place. Consequently, a general model is proposed for immunoglobulin differentiation. The hyperhomologous loci are postulated to be comprised of recombination sequences which makes them available for a mechanism of single-stranded DNA exposure. B cell maturation begins with V/C recombination, a step that is rate limiting. The fidelity of the process is ensured by extensive DNA homology between the two embryonic subgenes of V and C. Next, an error-prone repair system is activated and thereby introduces changes into the content of the immunoglobulin gene at the exposed loci. The process ends when mutations make the recombination sequence unrecognizable as such. The model is consistent with large amounts of data and is compatible with the view that immunoglobulin diversity is being generated somatically.
Serum stimulation of phospholipase A2 and prostaglandin release in 3T3 cells is associated with platelet-derived growth-promoting activity.
Authors: Shier W T
Published: May 2006

Sera from mouse, rat, and calf sources stimulate cellular phospholipase A2 activity (PLase; phosphatide 2-acylhydrolase, EC 3.1.1.4) and prostaglandin synthesis in 3T3 Swiss mouse fibroblasts, releasing up to 33% of biosynthetically incorporated [3H]arachidonic acid as hydrolysis products in 1 hr. The PLase stimulated by mouse serum exhibits specificity for arachidonic acid residues on phospholipids. It is stimulated 2.5-fold by 1.8 mM Ca2+ in the presence of 5 microM divalent cation ionophore A23187, consistent with a Ca2+-dependent enzyme possessing a cytoplasmic Ca2+ binding site. The percentage maximal PLase- and growth-stimulating activities of the three sera exhibit similar concentration dependencies, with the homologous (mouse) serum exhibiting the highest specific activities. Confluent 3T3 cells deplete PLase-stimulating activity from medium containing calf serum at a rate similar to the depletion of cell growth-stimulus activity. The PLase-stimulating activity in rat and mouse sera is derived from the leukocyte fraction of blood, presumably from platelets. In rat leukocyte lysates the PLase-stimulating activity exhibits properties similar to those reported for platelet-derived cell growth factors from rat and human sources--i.e., stability to exposure to 100 degrees C for 2 min or to pH 2 or pH 11, and cationic properties. Purified preparations of human platelet-derived growth factor also exhibit 3T3 PLase-stimulating activity.
Mitochondrial membrane biogenesis: identification of a precursor to yeast cytochrome c oxidase subunit II, an integral polypeptide.
Authors: Sevarino K A , Poyton R O
Published: May 2006

Many of the polypeptides made on endogenous ribosomes inside of yeast mitochondria are hydrophobic "integral polypeptides" which are subunits of at least three oligomeric enzyme complexes (cytochrome c oxidase, rutamycin-sensitive ATPase, and coenzyme QH2-cytochrome c reductase) of the inner mitochondrial membrane. In order to elucidate the pathway(s) followed by these polypeptides into the inner membrane we have used an in vitro mitochondrial translation system from yeast. By inhibiting this system with aurintricarboxylic acid, we have been able to demonstrate and accumulate a transient precursor to subunit II of cytochrome c oxidase. This precursor, designated II', is approximately 1,500 daltons larger than mature subunit II and most likely is a form of subunit II with an NH2-terminal extension. Although this precursor appears to be processed cotranslationally under normal conditions, it does associate in unprocessed form with mitochondrial membranes when allowed to accumulate in the presence of aurintricarboxylic acid, and it can be processed postranslationally upon removal of the drug. None of the other mitochondrial translation products made in this system exhibits larger precursors. These results indicate that at least one mitochondrial translation product has a transient "leader sequence" a,d is inserted into the inner mitochondrial membrane and processed cotranslationally, but they suggest that other pathways may be followed by the other translation products.
Antisickling activity of amino acid benzyl esters.
Authors: Gorecki M , Acquaye C T , Wilchek M , Votano J R , Rich A
Published: May 2006

The sickling of homozygous sickel cells upon deoxygenation is inhibited in the presence of 3 mM L-phenylalanine benzyl ester (Phe-OBzl) or benzyl esters of other aromatic or hydrophobic amino acids. Phe-OBzl was found to permeate into erythrocytes rapidly, and the deoxygenated cells maintained considerable flexibility as measured by their ability to pass through 3-micron pores. The osmotic fragility of the cells was unchanged and the oxygen dissociation curve was shifted slightly from a 50% saturation values of 28.5 mm Hg to 31.0 mm Hg. At lower concentrations of Phe-OBzl some antisickling activity was seen. The Phe-OBzl antisickling activity may involve both binding to deoxyhemoglobin S and modification of the erythrocyte membrane. This class of compounds has considerable potential as therapeutic agents for the treatment of sickle cell disease.
Arachidonate metabolism via lipoxygenase and 12L-hydroperoxy-5,8,10,14-icosatetraenoic acid peroxidase sensitive to anti-inflammatory drugs.
Authors: Siegel M I , McConnell R T , Porter N A , Cuatrecasas P
Published: May 2006

The enzymes of arachidonate metabolism via the lipoxygenase pathway in human platelet cytosol have been characterized and partially purified. The lipoxygenase activity has a pH optimum of 7.3 and reaches half-maximal activity at an arachidonate concentration of 80 microM. The oxidation of arachidonate by these enzymes is inhibited by reagents that modify sulfhydryl groups. Two separable lipoxygenase activities can be detected by chromatography of platelet cytosol on Sephadex G-150 and of partially purified preparations on DEAE-Sephadex. One of these has an apparent Mr of 100,000. A second enzyme species behaves as a Mr 160,000 entity containing, in addition to lipoxygenase, a peroxidase activity that catalyzes the conversion of 12L-hydroperoxy-5,8,10,14-icosatetraenoic acid (HPETE) to 12L-hydroxy-5,8,10,14-icosatetraenoic acid (HETE). Aspirin, indomethacin, sodium salicylate, phenylbutazone, ibuprofen, naproxen, and sulindac, but not acetaminophen or phenacetin, give rise to increased levels of HPETE in the lipoxygenase pathway. This increase in HPETE levels is the result of the ability of these drugs to inhibit directly the enzymatic conversion of HPETE to HETE.
Epidermal growth factor and expression of specific genes: effects on cultured rat pituitary cells are dissociable from the mitogenic response.
Authors: Johnson L K , Baxter J D , Vlodavsky I , Gospodarowicz D
Published: May 2006

Cultured rat pituitary tumor cells, GH3/D6, which synthesize both growth hormone and prolactin, have cell-surface epidermal growth factor (EGF) receptor sites (34,000 per cell) that bind 125I-labeled EGF with a high affinity (Kd approximately 1 nM). Prolonged treatment of the cells with EGF did not stimulate cell division but did inhibit thyroid hormone-stimulated cell growth. In addition, EGF altered the morphology of the cells from a rounded to an elongated conformation. EGF also induced a perturbation of chromatin structure in GH3 cell nuclei that was detected by an increase (40%) in the number of rifampicin-resistant initiation sites for bacterial RNA polymerase. This was accompanied by an increased synthesis of prolactin and an inhibition of synthesis of growth hormone. In the presence of EGF, the synthesis of growth hormone was no longer inducible by thyroid hormone, but it remained responsive to glucocorticoids. The results demonstrate that EGF can elicit major effects on the cellular phenotype and expression of specific genes in the absence of a proliferative response. This suggests that EGF can also regulate differentiated cellular functions.
Induction of differentiation of human and murine myeloid leukemia cells in culture by tunicamycin.
Authors: Nakayasu M , Terada M , Tamura G , Sugimura T
Published: May 2006

Tunicamycin, an antibiotic that specifically blocks the synthesis of N-acetylglucosamine-lipid intermediates and thereby prevents glycosylation of glycoproteins, induced differentiation of both human (HL-60) and murine (M1) myeloid leukemia cell lines in culture. At 0.1-1.0 microgram/ml, it induced differentiation of both HL-60 and M1 cells, characterized by increase in phagocytic cells and changes to resemble mature myeloid cells. Fc receptors were also induced in M1 but not in HL-60 cells; induction of intracellular lysozyme activity was not detected in either HL-60 or M1 cells. With this concentration of tunicamycin, there was marked decrease in rate of incorporation of radioactive glucosamine into macromolecules and a decrease in the rate of DNA synthesis. These data show that glycosylation of cellular proteins has an important role in maintaining these myeloid leukemia cells in an undifferentiated state in culture. The results also indicate that induction of phagocytosis in both HL-60 and M1 myeloid leukemia cells and of Fc receptors in M1 cells does not require continued synthesis of the oligosaccharide portions of cellular proteins by the lipid-linked pathway.
Triplicated alpha-globin loci in humans.
Authors: Goossens M , Dozy A M , Embury S H , Zachariades Z , Hadjiminas M G , Stamatoyannopoulos G , Kan Y W
Published: May 2006

We have identified 12 individuals who are heterozygous for a chromosome with three alpha-globin genes. We determined the presence of the third alpha-globin locus by restriction endonuclease digestion and hybridization with alpha-globin cDNA probes. The three alpha-globin loci resided in an elongated fragment on digestion with EcoRI, BamHI, and Xba I, and the third locus was present in an additional 3.7-kilobase fragment on digestion with Hpa I, Sac I, and Bgl II. The locations of the restriction sites are compatible with a nonhomologous crossover mechanism producing the triple alpha loci. The frequency of the triple alpha loci was 0.0036 in American blacks and less than 0.004 in Sardinians, but was higher in Greek Crypriots (0.05). Individuals with five alpha-globin genes (alpha alpha/alpha alpha alpha) apparently showed no clinical or hematologic abnormalities.
Evidence for an important physiological role for calcitonin
Authors: Talmage Roy V. , Grubb Stephen A. , Norimatsu Hirotashi , VanderWiel Carole J.
Published: May 2006

We propose that calcitonin, secreted in response to the intake of food, aids in routing calcium, obtained by intestinal absorption, into bone fluid. Here calcium is temporarily stored in combination with phosphate for return to the extracellular fluid (blood) during intervals between oral intakes of calcium. The net result is a conservation of calcium postprandially and a decrease in parathyroid hormone-induced bone destruction during subsequent fasting periods. Evidence for this postulate is provided in the following six sequential steps from the time a calcium-containing meal is consumed until that portion of calcium stored in bone fluid is utilized during fasting periods to aid in plasma calcium maintenance. (
i
) Calcitonin secretion is stimulated by feeding and subsequent digestive processes. (
ii
) Postprandial secretion of calcitonin restricts the efflux of calcium from bone fluid to blood, thereby maintaining parathyroid hormone (PTH) secretion. (
iii
) In thyroid-intact individuals, both PTH and calcitonin are secreted postprandially and act in concert on calcium homeostasis. (
iv
) Calcitonin actively moves phosphate into bone and prevents its loss from bone fluid to blood. (
v
) Postprandial storage of calcium with phosphate occurs in bone fluid of thyroid-intact individuals. (
vi
) This labile storage form of calcium is the first to be utilized during fasting periods. In the absence of partial disruption of this storage mechanism, rapid development of pathological bone conditions would
not
be expected because PTH action permits the extended utilization of bone calcium for plasma calcium control. However, augmentation of osteopenic conditions could be expected if long-term low calcium intake were accompanied by a malfunction of this calcitonin-induced system for calcium storage.
Substance P enhances cholinergic receptor desensitization in a clonal nerve cell line.
Authors: Stallcup W B , Patrick J
Published: May 2006

Substance P inhibits carbamylcholine-induced 22Na+ uptake in the clonal cell line PC12. This inhibition is noncompetitive with agonist but competitive with Na+. Octahydrohistrionicotoxin (H8-HTX) also exhibits this same pattern of inhibition. Moreover, both substance P and H8-HTX are very effective in enhancing agonist-induced receptor desensitization. Local anesthetics, such as QX222, also cause inhibition that is competitive with Na+, but they have only marginal effects on desensitization. Because substance P and H8-HTX cannot by themselves cause desensitization, their action is dependent on and synergistic with the action of agonist. Furthermore, substance P and H8-HTX do not appear to compete for the same site as QX222, which is thought to bind to the ion channel. Finally, substance P can stabilize the desensitized state of the receptor even when added subsequent to the actual desensitization and removal of agonist. Thus, substance P does not require open ion channels for binding and may modulate the activity of the receptor-ionophore complex by binding to a distinct regulatory site.
5-fluorouracil-methotrexate synergy: enhancement of 5-fluorodeoxyridylate binding to thymidylate synthase by dihydropteroylpolyglutamates.
Authors: Fernandes D J , Bertino J R
Published: May 2006

Ternary complex formation of thymidylate synthase (5,10-methylenetetrahydrofolated:dUMP C-methyltransferase, EC 2.1.1.45), 5-fluorodeoxyuridylate (FdUMP), and poly(gamma-glutamyl) conjugates of pteroate and methotrexate (MTX) has been examined as a basis for the sequence-dependent synergism of the 5-fluorouracil-MTX combination in inhibiting viability of L1210 murine tumor cells. A 1.4-log (25-fold) increase in the inhibition of soft agar colony formation was observed when MTX preceded 5-fluorouracil as compared to the reverse sequence. L1210 cells converted 39% of the total intracellular MTX into MTX poly(gamma-glutamate)s within 4 hr of exposure to 1 microM MTX. MTX and MTX(gamma-glutamate) formed reversible ternary complexes with FdUMP on one site of thymidylate synthase, whereas with 7,8-dihydropteroylpentaglutamate and I-5,10-methylenetetrahydropteroylpentaglutamate stoichiometric binding of FdUMP to two sites on thymidylate synthase was observed. The dissociation constants for FdUMP in the ternary complexes formed in the presence of MTX, MTX(gamma-glutamate), 7,8-dihydropteroylpentaglutamate, and I-5-10-methylenetetrahydropteroylpentaglutamate were estimated to be 370, 27, < 10, and < 10 nM, respectively, by equilibrium dialysis. We propose that the sequence-dependent effect of MTX plus 5-fluorouracil on L1210 cell viability results from MTX and MTX polyglutamate inhibition of dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) and consequently a trapping of intracellular folates as dihydropteroylpolyglutamates, which increase the extent of FdUMP binding to thymidylate synthase.
Cloning and partial nucleotide sequence of human immunoglobulin mu chain cDNA from B cells and mouse-human hybridomas.
Authors: Dolby T W , Devuono J , Croce C M
Published: May 2006

Purified mRNAs coding for mu and kappa human immunoglobulin polypeptides were translated in vitro and their products were characterized. The mu-specific mRNAs, derived from both human lymphoblastoid cells (GM607) and from a mouse-human somatic cell hybrid secreting human mu chains (alpha D5-H11-BC11), were copied into cDNAs and inserted into the plasmid pBR322. Several recombinant cDNAs that were obtained were identified by a combination of colony hybridization with labeled probes, in vitro translation of plasmid-selected mu mRNAs, and DNA nucleotide sequence determination. One recombinant DNA, for which the sequence has been partially determined, contains the codons for part of the C3 constant region domain through the carboxy-terminal piece (155 amino acids total) as well as the entire 3' noncoding sequence up to the poly(A) site of the human mu mRNA. The sequence A-A-U-A-A occurs 12 nucleotides prior to the poly(A) addition site in the human mu mRNA. Considerable sequence homology is observed in the mouse and human mu mRNA 3' coding and noncoding sequences.
Synthesis and biological actions of prosomatostatin.
Authors: Meyers C A , Murphy W A , Redding T W , Coy D H , Schally A V
Published: May 2006

The recently isolated 28-residue sequence of prosomatostatin, a putative somatostatin precursor from pig hypothalamus and intestine, was synthesized by solid-phase methodology, characterized, and tested in rats for its effects on the release of insulin, glucagon, growth hormone, and prolactin. The synthetic product strongly suppressed plasma levels of insulin, glucagon, and growth hormone, and it appeared to be more active in the pancreas than in the pituitary. It inhibited insulin release about 5 times more effectively than somatostatin on a weight basis. The prohormone also suppressed growth hormone and prolactin levels in vitro. A time-course experiment for the effect of prosomatostatin on growth hormone release in vivo showed a significant suppression of plasma growth hormone for at least 90 min.