bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of pyruvate dehydrogenase by direct addition of insulin to an isolated plasma membrane/mitochondria mixture: Evidence for generation of insulin's second messenger in a subcellular system
Authors: Seals Jonathan R. , Jarett Leonard
Published: May 2006

The addition of insulin to a mixture of plasma membrane and mitochondrial fractions from rat adipocytes results in a decrease in the phosphorylation of a mitochondrial protein identified as the α subunit of pyruvate dehydrogenase [pyruvate:lipoamide oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1] (Seals, J. R., McDonald, J. M. & Jarett, L. (1979)
J. Biol. Chem.
254, 6991-6996). This study confirms the prediction that a corresponding increase in pyruvate dehydrogenase activity can be effected by insulin treatment of this preparation. Incubation of the plasma membrane/mitochondria mixture with ATP inhibited pyruvate dehydrogenase activity as measured in a subsequent enzyme assay. The presence of insulin during this incubation with ATP resulted in a 24.5% stimulation of enzyme activity compared to incubation without insulin (
n
= 9,
P
< 0.001). The effect was specific for biologically active insulin and was insulin dose-dependent in the physiological range of insulin. Supermaximal doses of insulin produced reduced effects. An insulin effect of similar magnitude could also be observed when the plasma membrane/mitochondria mixture was incubated without ATP. Two insulin mimickers, concanavalin A and antibody to insulin receptor, stimulated pyruvate dehydrogenase by 30.4% (
n
= 6,
P
<0.001) and 28.1% (
n
= 8,
P
<0.001), respectively. Both of these agents also produced reduced effects at supermaximal concentrations. The effects of all three agents required plasma membranes and could not be produced by treatment of mitochondria alone. The results suggest that a mechanism common to all three agents is responsible for transmitting the stimulation from the plasma membrane to the mitochondrial components of the mixture.
Microwave spectrum and molecular structure of the carbon monoxide-hydrogen bromide molecular complex
Authors: Keenan M. R. , Minton T. K. , Legon A. C. , Balle T. J. , Flygare W. H.
Published: May 2006

Rotational spectra have been assigned for five isotopic species of the OC···HBr hydrogen-bonded molecular complex by using pulsed microwave Fourier transform spectroscopy in a Fabry-Perot cavity and a pulsed supersonic nozzle as the molecular source. The rotational constants, centrifugal distortion constants, Br nuclear-quadrupole-coupling constants, and Br spin-rotation constants were determined and from them, the vibrationally averaged structure of OCHBr was derived. This structure is consistent with a linear geometry at equilibrium and an atomic arrangement as written above. The intermolecular potential binding CO to HBr is discussed.
4-Norleucine, 7-D-phenylalanine-alpha-melanocyte-stimulating hormone: a highly potent alpha-melanotropin with ultralong biological activity.
Authors: Sawyer T K , Sanfilippo P J , Hruby V J , Engel M H , Heward C B , Burnett J B , Hadley M E
Published: May 2006

alpha-Melanocyte-stimulating hormone (alpha-MSH) reversibly darkens frog skins by stimulating melanosome movement (dispersion) within melanophores. Heat-alkali treatment of alpha-MSH results in prolonged biological activity of the hormone. Quantitative gas chromatographic analysis of the hydrolyzed heat-alkali-treated peptide revealed partial racemization particularly at the 4(methionine) and 7(phenylalanine) positions. [Nle4]-alpha-MSH, a synthetic analogue of alpha-MSH, reversibly darkens frog skins and also exhibits prolonged activity after heat-alkali treatment. Synthesis of [Nle4, D-Phe7]-alpha-MSH provided an analogue with prolonged biological activity identical to that observed with heat-alkali-treated alpha-MSH or [Nle4]-alpha-MSH. [Nle4, D-Phe7]-alpha-MSH was resistant to enzymatic degradation by serum enzymes. In addition, this peptide exhibited dramatically increased biological activity as determined by frog skin bioassay, activation of mouse melanoma adenylate cyclase, and stimulation of mouse melanoma cell tyrosinase activity. This Nle4, D-Phe7 synthetic analogue of alpha-MSH is a very porent melanotropin, 26 times as potent as alpha-MSH in the adenylate cyclase assay. The resistance of the peptide to enzymatic degradation and its extraordinarily potent and prolonged biological activity should make this analogue of alpha-MSH an important molecular probe for studying the melanotropin receptors of both normal and abnormal (melanoma) melanocytes.
Insulin and serum increase the number of receptors for vasopressin in a kidney-derived line of cells grown in a defined medium.
Authors: Roy C , Preston A S , Handler J S
Published: May 2006

A continuous strain of epithelial cells (LLC-PK1L) derived initially from pig kidney grows in a defined medium without serum or hormones. The epithelia from domes, a manifestation of transepithelial transport. The parent cell line, LLC-PK1, forms similar eithelia and has vasopressin-sensitive adenylate cyclase. In this study the binding of 3H-labeled [Lys8]vasopressin was used to examine the vasopressin receptors of LLC-PK1L cells. In the absence of serum, LLC-PK1L cells have receptors with a Kd (10 nM) for 3H-labeled [Lys8]vasopressin similar to that of the parent cell line and of membranes prepared from pig renal medulla, LLC-PK1L cells, however, have only 5% as many receptors as LLC-PK1 cells. The number of receptors and the vasopressin-sensitive adenylate cyclase activity are increased by growing LLC-PK1L cells in medium with 10% fetal bovine serum or with 10 microgram of insulin per ml. The combination of serum plus insulin has a greater effect than either alone. Serum and insulin do not change the apparent affinity of the receptors for vasopressin or the coupling between receptors and adenylate cyclase. Basal and NaF-stimulated adenylate cyclase activity are not affected. Several agents reported to induce receptors for hormones in other tissues have no effect on vasopressin receptors in LLC-PK1L cells.
Growth of T-lymphoma cells in serum-free medium: lack of involvement of the cyclic AMP pathway in long-term cultures.
Authors: Darfler F J , Murakami H , Insel P A
Published: May 2006

We have developed a serum-free, chemically defined growth medium containing casein, insulin, transferrin, testosterone, and linoleic acid in Dulbecco's modified Eagle's medium/Ham's F12 medium, 1:1 (vol/vol), for growing murine T lymphomas. This medium supports the growth in suspension of all murine T lymphomas tested, including S49, WEHI 7, EL4, BW5147, and R1.1. Growth of these cell lines was maintained indefinitely with doubling times approaching those of cells grown in 10% (vol/vol) horse serum. This medium also supports the growth of several of the S49 variants of the beta-adrenergic receptor/adenylate cyclase/cyclic AMP/protein kinase pathway, suggeting little or no involvement of this pathway in the routine growth of S49 cells or in the mechanism of action of the factors in this defined medium. This serum-free medium should prove useful for studies of a variety of metabolic pathways and of differentiated functions of T-lymphoma cells.
Regulation of the hexosamine biosynthetic pathway in the water mold
Blastocladiella emersonii:
Sensitivity to endproduct inhibition is dependent upon the life cycle phase
Authors: Selitrennikoff Claude P. , Dalley Norman E. , Sonneborn David R.
Published: May 2006

Chitin, a homopolymer of
N
-acetylglucosamine (GlcNAc), is the major macromolecular constituent of
Blastocladiella emersonii
cell walls. Zoospores do not possess a wall nor do they contain sufficient total hexosamine to account for the chitin content of the wall abruptly formed during germination. UDPGlcNAc, both the endproduct of hexosamine biosynthesis and the substrate for chitin synthesis, is present in zoospores in sufficient concentration to inhibit the first hexosamine pathway-specific enzyme activity. Net chitin accumulates in register with dry weight during exponential growth, but does not accumulate appreciably during the succeeding sporulation phase. Predicted relationships among net rates of chitin synthesis, UDPGlcNAc concentrations, and UDP plus UTP concentrations throughout the life cycle are explored, as are the assumptions upon which the predictions were based. We find that the sensitivity of the first hexosamine pathway-specific enzyme to endproduct inhibition is
not
constant throughout the life cycle; sensitivity is very high in the zoospore phase, decreases dramatically during germination, remains very low through the growth phase, and increases gradually to the zoospore level during sporulation. The organism appears to have evolved endproduct regulation in this case as an adaptation to “hard-times” phases of the life cycle—i.e., as a safeguard against overproduction of end product (UDPGlcNAc) when its utilization in cell wall (specifically chitin) synthesis is curtailed. Conversely, the organism effectively discards this mode of regulation during “good times,” when the demands for end product are evidently greater than endproduct inhibition would otherwise permit.
Evidence for unequal crossing over within the mouse T/t complex.
Authors: Silver L M , White M , Artzt K
Published: May 2006

The Tcp-1 gene located within the T/t complex on chromosome 17 of the mouse codes for a major cell surface-associated protein p63/6.9. Previously, we identified two structural alleles of this gene which specify alternate forms of the p63/6.9 protein. The Tcp-1b allele is associated with all wild-type chromosome 17; the Tcp-1a allele is found only with chromosome 17 carrying a complete t haplotype. Normal recombination along a major length of chromosome 17 is suppressed in mice that are heterozygous for any complete t haplotype. Suppression is not complete, however, and rare crossing over between wild-type and t haplotype chromatin does occur. In this report, 15 rare recombinant chromosomes have been analyzed for Tcp-1 alleles. The results indicate that in four independent events the Tcp-1b and Tcp-1a alleles have become associated in cis position in a single DNA molecule. Further genetic analysis provides support for the hypothesis that a significant nonhomology exists between the arrangement of DNA sequences on wild-type and t-carrying chromosome 17. This could account for both the suppression of normal recombination along the stretch of t chromatin and the frequent unequal crossing over when rare recombinational events do take place.
ATP depletion as a consequence of adenosine deaminase inhibition in man.
Authors: Siaw M F , Mitchell B S , Koller C A , Coleman M S , Hutton J J
Published: May 2006

Hereditary deficiency of the enzyme adenosie deaminase (adenosine aminohydrolase, EC 3.5.4.4) results in an immunodeficiency syndrome characterized by a marked reduction in circulating lymphocytes. We have administered 2'-deoxycoformycin, a potent inhibitor of adenosine deaminase, to a patient with a lymphoproliferative malignancy. The clinical consequences of pharmacologic inhibition of adenosine deaminase activity included an abrupt decrease in the lymphocyte count, abnormalities of renal and hepatic function, and hemolytic anemia. The plasma concentrations of adenosine and deoxyadenosine rose to peak values of 13 microM and 5 microM, respectively, and erythrocyte dATP levels increased to 110 pmol/10(6) cells over 9 days. There was a corresponding decrease in erythrocyte ATP levels from 128 to < 6 pmol/10(6) cells. A similar profound reductin in ATP occurred in the erythrocytes of a second patient. The rapid and unexpected depletion of ATP associated with dATP accumulation may account, at least in part, for the toxicity associated with 2'-deoxycoformycin administration. The inverse relationship of ATP and dATP raises major questions about the control of energy metabolism in erythrocytes.
Specific stimulated uptake of acetylcholine by Torpedo electric organ synaptic vesicles.
Authors: Parsons S M , Koenigsberger R
Published: May 2006

The specificity of acetylcholine uptake by synaptic vesicles isolated from the electric organ of Torpedo californica was studied. In the absence of cofactors, [3H]acetylcholine was taken up identically to[14C]choline in the same solution (passive uptake), and the equilibrium concentration achieved inside the vesicles was equal to the concentration outside. In the presence of MgATP, [3H]acetylcholine and [14C]choline in the same solution were taken up identically, except only about half as much of each was taken up (suppressed uptake). [3H]Acetylcholine uptake was stimulated by MgATP and HCO3- about 4-fold relative to suppressed uptake, for a net concentrative uptake of about 2:1 (stimulated uptake). Uptake of [14C]choline in the same solution remained at the suppressed level. [3H]Acetylcholine taken up under stimulated conditions migrated with vesicles containing [14C]mannitol on analytical glycerol density gradients during centrifugation. Vesicle were treated with nine protein modification reagents under mild conditions. Two reagents had no effect on, dithiothreitol potentiated, and six reagents strongly inhibited subsequent stimulated uptake of [3H]acetylcholine. The results indicate that uptake of acetylcholine is conditionally specific for the transported substrate, is carried out by the synaptic vesicles rather than a contaminant of the preparation, and requires a functional protein system containing a critical sulfhydryl group.
Analytic construction of Hartree-Fock density matrices
Authors: Payne Philip W.
Published: May 2006

A method is presented for variational calculation of the energy and the spin densities derived from a single-determinant wavefunction. Sum and difference coordinates [unk]
R
= ½([unk]
r
1
+ [unk]
r
1
) and [unk]
r
= [unk]
r
1
- [unk]
r
1
are introduced, and the density matrix
P
([unk]
r
1
,[unk]
r
1
) is expanded in partial waves in the new coordinate frame: [Formula: see text] The functions

h
L

(ε,
r
) are bound or continuum hydrogenic functions with energy ε.

It is shown that the spin densities depend on the
s
partial waves only, and a Euler equation for these partial waves is derived: [Formula: see text] in which
U
([unk]
R
) is the electrostatic potential,
a
(ε) =
h
0
(ε,0), and μ is chosen to normalize the spin density to
N
electrons. Further, the electronic energy can be expressed in terms of the
s
partial waves and the constant μ in the above equations.

The idempotent density matrix that ensues from a particular choice of functions {
B
00
[unk](ε,[unk]
R
)} is generated by choosing partial density waves {

B
LM

(ε,[unk]
R
),
L
> 0} so that tr[
P
2
(1 -
P
)
2
] is minimized.
A two-reservoir recycling model for mantle-crust evolution
Authors: Jacobsen S. B. , Wasserburg G. J.
Published: May 2006

The exact solutions for the isotopic compositions and the concentrations of the two-reservoir model for mantle-crust evolution are given for arbitrary rates of crustal growth and of back flow to the mantle. The critical parameters are the chemical fractionation factors for crustal growth and refluxing and the integrated fractional mass-removal rates from the crust and the mantle. For the case where refluxing is proportional to crustal growth, all the solutions reduce to simple analytic expressions. The expression for the mean age of the mass of the crust with refluxing is given. If refluxing is significant, the model shows that highly incompatible elements have short residence times in the mantle. With plausible concentration values, material balance implies that the continents were derived from only a small fraction of the mantle.
Mitochondrial DNA polymorphism: evidence that variants detected by restriction enzymes differ in nucleotide sequence rather than in methylation.
Authors: Castora F J , Arnheim N , Simpson M V
Published: May 2006

Restriction enzyme analysis of mtDNAs for the purpose of determining sequence divergence rests on the assumption that variant recognition sites differ with respect to sequence and not methylation. This assumption was tested on two mtDNAs, A and B, which are distributed throughout the laboratory rat population and which can be distinguished by a number of restriction enzymes. The mtDNAs were cloned and the nucleotide sequences of corresponding small HindIII fragments, in which a variant EcoRI site occurs, were determined. Evidence that the fragments differ in sequence and not methylation is as follows: (i) The cloned mtDNA yielded the same fragment pattern as did native mtDNA when treated with EcoRI, Hha I, HinfI, and Hae III; (ii) three nucleotide replacements were found in the 169-base pair fragment, A.T in equilibrium G.C, A.T in equilibrium G.C, T.A in equilibrium G.C; (iii) one of these replacements, A.T in equilibrium G.C at position 80, accounts for the presence of the EcoRI site in the type A and its absence in the type B mtDNA. Examination of the sequence leads to the suggestion that these three nucleotide replacements are silent; i.e., they would not lead to amino acid substitutions in a possible encoded protein.
Structure and multiplicity of genes for the human immunoglobulin heavy chain variable region.
Authors: Matthyssens G , Rabbitts T H
Published: May 2006

Several genes for the variable region of immunoglobulin heavy chains (VH genes) have been isolated from human fetal liver DNA by using a cDNA plasmid probe containing a mouse VH sequence. The detectable VH genes are separated by 12-16 kilobases of DNA, and hybridization experiments show about 23 hybridizing VH genes in DNA of three different individuals. The complete nucleotide sequence of one of these human VH genes shows that it belongs to the human VHIII subgroup. The VH gene appears to contain an intervening sequence (104 bases in length) within a precursor sequence, between residues -4 and -5. The precursor sequence is itself 19 codons in length. The 3' end of the V gene seems to be at codon 93 or 94, and this is followed by the conserved sequences C-A-C-A-G-T-G and G-A-C-A-C-A-A-A-C-C. The presence of these sequences suggests that similar enzymatic mechanisms are involved in the integration of V genes in both heavy and light chains.
Induction of growth in kidney epithelial cells in culture by Na+.
Authors: Toback F G
Published: May 2006

The role of Na+ in the regulation of cell growth was examined in quiescent, high-density cultures of kidney epithelial cells of the BSC-1 line. The addition of NaCl to the medium increased the number of cells initiating DNA synthesis in a concentration-dependent manner after serum stimulation. In the presence of added NaCl, cells in confluent cultures grew to high density at an increased rate, whereas growth in sparse cultures was retarded. These results suggest that, in the presence of serum, Na+ can act as a mediator of the events that initiate cell proliferation.
Synthesis of a posterior indicator protein in normal embryos and double abdomens of Smittia sp. (Chironomidae, Diptera).
Authors: Jäckle H , Kalthoff K
Published: May 2006

In embryos of the chironomid midge Smittia, synthesis of a posterior indicator protein designated PI1 (Mr approximately 50,000; pI approximately 5.5) forecasts development of an abdomen as opposed to head and thorax. The protein is synthesized several hours before germ anlage formation. In normal embryos at early blastoderm stages, synthesis of PI1 is restricted to posterior embryonic fragments but not to pole cells. In "double-abdomen" embryos, a mirror-image duplication of the abdomen is formed by cells that would otherwise develop into head and thorax. Embryos were programmed for double-abdomen development by UV irradiation of the anterior pole, and half of them were reprogrammed for normal development by subsequent exposure to visible light (photoreversal). Correspondingly, PI1 was synthesized in anterior fragments of UV-irradiated embryos but not after photoreversal. In a control experiment, UV irradiation of the posterior pole caused neither double-abdomen formation nor PI1 synthesis in anterior fragments. The identity of PI1 formed in anterior fragments of prospective double abdomens with the protein found in posterior fragments was revealed by two-dimensional gel electrophoresis and limited proteolysis. Suppression of PI1 synthesis in anterior fragments of normal embryos is ascribed to the activity of cytoplasmic ribonucleoprotein particles thought to act as anterior determinants.
Synaptonemal complexes at premeiotic interphase in the mouse spermatocyte.
Authors: Grell R F , Oakberg E F , Generoso E E
Published: May 2006

Male mice were injected intraperitoneally with 125 microCi (1 Ci = 3.7 X 10(10) becquerels) of [3H]thymidine at 1-hr intervals and killed 1 hr after the second injection. Testes were prepared for bright-field and electron microscopic autoradiography. Primary spermatocytes, identified by light microscopy to be at the premeiotic interphase stage, were found to be heavily labeled. Electron microscopic examination disclosed the coincidental occurrence of synaptonemal complexes and label within the nuclei of premeiotic interphase spermatocytes, indicating synapses of homologues had begun during the S phase. The significance of this finding for the traditional view of meiosis is discussed.
Intermolecular anti-parallel β sheet: Comparison of predicted and observed conformations of gramicidin S
Authors: Rackovsky S. , Scheraga H. A.
Published: May 2006

A recently determined x-ray structure of the hydrated gramicidin
S
-urea complex is compared with a structure predicted by conformational energy minimization. It is shown that the two structures are in good general agreement, including the prediction of a hydrogen bond between the side-chain amino group of ornithine and the backbone carbonyl of phenylalanine. This agreement demonstrates the power of empirical potential energy methods in conformational analysis and illustrates one method for solution of the multiple-minimum problem for small peptides. It is noted that, in the crystal, gramicidin
S
is a dimer that forms an
intermolecular
antiparallel four-stranded β sheet and that differences between the predicted and x-ray structures can be explained by this intermolecular interaction. The residual conformational asymmetry of the x-ray structure is shown to be due to the formation of the complex with urea.
Macromolecular photoaffinity labeling of the lutropin receptor on granulosa cells.
Authors: Ji I , Ji T H
Published: May 2006

Lutropin, a pituitary hormone, and human choriogonadotropin bind to the same receptors in the ovary and elicit identical responses. A photoactivable derivative of human choriogonadotropin was used to identify the lutropin receptor on porcine granulosa cells. The hormone was condensed with a heterobifunctional reagent, the N-hydroxysuccinimide ester of 4-azidobenzoylglycylglycine, and iodinated. The 125I-labeled hormone (125I-hormone) derivative associated with the same number of receptors as 125I-hormone itself did but with a slightly lower Ka, 2.98 X 10(9) M-1 compared with 5.1 X 10(9) M-1 for 125I-hormone. The binding could be blocked with untreated hormone or lutropin but not with follitropin, prolactin, insulin, or bovine serum albumin. Its alpha and beta subunits could be crosslinked to produce alpha beta dimer by photolysis, the extent of crosslinking being dependent upon the reagent concentration used for the derivatization: 22.8% at 50 microM, 37.3% at 100 microM, and 67.2% at 150 microM. When the 125I-hormone derivative bound to the cells was photolyzed for crosslinking and the products resolved by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels under reducing conditions, three new bands of lower electrophoretic mobility appeared in addition to alpha, beta, and alpha beta bands. Formation of these crosslinked complexes required photolysis and the presence of both cells bearing the receptor and the 125I-hormone derivative. It could be blocked by excess untreated hormone. The three bands correspond to molecular weights, 96,000 +/- 6,700, 66,000 +/- 4,600, and 63,000 +/- 4,400. Because the hormone has a high carbohydrate content and such glycoproteins are known to exhibit anomalous electrophoretic mobilities, these estimates must be tentative.
Genetic and DNA sequence analysis of the kanamycin resistance transposon Tn903.
Authors: Grindley N D , Joyce C M
Published: May 2006

The kanamycin resistance transposon Tn903 consists of a unique region of about 1000 base pairs bounded by a pair of 1050-base-pair inverted repeat sequences. Each repeat contains two Pvu II endonuclease cleavage sites separated by 520 base pairs. We have constructed derivatives of Tn903 in which this 520-base-pair fragment is deleted from one or both repeats. Those derivatives that lack both 520-base-pair fragments cannot transpose, whereas those that lack just one remain transposition proficient. One such transposable derivative, Tn903 delta I, has been selected for further study. We have determined the sequence of the intact inverted repeat. The 18 base pairs at each end are identical and inverted relative to one another, a structure characteristic of insertion sequences. Additional experiments indicate that a single inverted repeat from Tn903 can, in fact, transpose; we propose that this element be called IS903. To correlate the DNA sequence with genetic activities, we have created mutations by inserting a 10-base-pair DNA fragment at several sites within the intact repeat of Tn903 delta 1, and we have examined the effect of such insertions on transposability. The results suggest that IS903 encodes a 307-amino-acid polypeptide (a "transposase") that is absolutely required for transposition of IS903 or Tn903.
Vanadate inhibition of auxin-enhanced H
+
secretion and elongation in pea epicotyls and oat coleoptiles
Authors: Jacobs Mark , Taiz Lincoln
Published: May 2006

In experiments carried out to investigate the acid secretion theory of auxin action, we utilized sodium orthovanadate, an agent found to be a selective inhibitor of a plasma membrane-associated H
+
-pumping ATPase in
Neurospora
[Bowman, B. J. & Slayman, C. W. (1979)
J. Biol. Chem.
245, 2928-2934]. At 1 mM, vanadate inhibited auxin-enhanced medium acidification by pea epicotyl segments within 5 min, whether added 0.5 or 2.5 hr after auxin. Inhibition of acidification was total after 10-15 min but could be reversed within 10 min after vanadate removal. When given as a 40-min pretreatment, vanadate completely prevented any auxin-enhanced acidification. Vanadate inhibition of medium acidification by oat coleoptile segments was also total and reversible, but both inhibition and reversal occurred after longer lag times than in pea. Inhibitory effects of vanadate on elongation in pea and oat tissue closely paralleled its effects on acidification, and the inhibitory effect of vanadate on elongation could be reversed by an acidic buffer. Vanadate did not inhibit respiration or protein synthesis in pea epicotyl segments, although it strongly inhibited L-[
14
C]leucine uptake. These results indicate the importance of cell wall acidification for short- and long-term auxin-enhanced growth and suggest the participation in wall acidification of a plasma membrane-associated ATPase acting as an H
+
pump.
Receptor-mediated transcellular transport of immunoglobulin: synthesis of secretory component as multiple and larger transmembrane forms.
Authors: Mostov K E , Kraehenbuhl J P , Blobel G
Published: May 2006

We have characterized the early biosynthetic forms of secretory component (SC). SC is synthesized in various glandular epithelial cells and functions in transepithelial transport of certain polymeric immunoglobulins. In rabbit, mature SC is known to be heterogeneous, consisting of two or three immunologically related glycoproteins. Using translation of mRNA from rabbit mammary gland or liver in a wheat germ cell-free system supplemented with dog pancreas microsomal vesicles, we discovered that the translation products of SC mRNA include at least four distinct polypeptides. Moreover, we found that all four polypeptides are synthesized not as soluble secretory forms but as considerably larger transmembrane forms that are core-glycosylated and asymmetrically integrated into the dog pancreas microsomal vesicles in a translation-coupled manner. We therefore conclude that the mature secreted forms of SC are endoproteolytic fragments derived from and comprising the ectoplasmic portion of the transmembrane precursor forms. The detection of transmembrane precursor forms for mature SC provides a protein structural basis for their function as receptors mediating transepithelial transport of immuno-globulin molecules.
Calmodulin activation of plant microsomal Ca
2+
uptake
Authors: Dieter Peter , Marmé Dieter
Published: May 2006

The active ATP-dependent uptake of Ca
2+
into a plasma membrane-enriched microsomal fraction from plants is stimulated by the addition of plant as well as bovine brain calmodulin. This stimulation occurs only when the microsomes are prepared in the presence of EDTA. Plant calmodulin also activates the bovine brain 3′,5′-cyclic-nucleotide phosphodiesterase (EC 3.1.4.17). Mitochondrial Ca
2+
accumulation is not affected by calmodulin. The calmodulin-mediated activation of microsomal Ca
2+
uptake is abolished by the antipsychotic drug fluphenazine, whereas the calmodulin-independent Ca
2+
uptake is only slightly decreased.
Molecular analysis of a gene in a developmentally regulated puff of Drosophila melanogaster.
Authors: Muskavitch M A , Hogness D S
Published: May 2006

An increase in the concentration of the steroid hormone ecdysone in late larval life triggers a profound change in the pattern of polytene chromosome puffs in the Drosophila melanogaster salivary gland. One of the preexisting puffs that regress as the ecdysone concentration increases is located at the 3C11-12 bands, the site of the Sgs-4 gene, which codes for the sgs-4 protein, one of the proteins in the salivary glue secretion. We have isolated cloned segments of chromosomal DNA that define a 60-kilobase region containing the 0.9-kilobase Sgs-4 gene, and we have determined its position and orientation within this region. Fine structure restriction endonuclease mapping shows that approximately 45% of this gene consists of tandemly repeated sequences of 21 base pairs that occupy most of its 5' half, indicating that most of the amino-terminal half of the sgs-4 protein consists of tandemly repeated amino acid sequences of seven residues. We also report on the amount of the Sgs-4 mRNA as a function of developmental stage and in nine different strains, four of which produce little or no sgs-4 protein. Three of the null strains produce minute amounts of the mRNA and one yields none, whereas the five sgs-4 producing strains yield abundant amounts. The mRNAs frm these strains exhibit different lengths, which correlate with different gene lengths that appear to result from different numbers of the repeated sequences in their tandem arrays.
H-2 restriction as a consequence of intentional priming: T cells of fully allogeneic chimeric mice as well as of normal mice respond to foreign antigens in the context of H-2 determinants not encountered on thymic epithelial cells.
Authors: Stockinger H , Pfizenmaier K , Hardt C , Rodt H , Röllinghoff M , Wagner H
Published: May 2006

Fully allogeneic chimeras were able to develop in vitro alloantigen-specific, as well as H-2-restricted, Sendai virus-specific cytotoxic T-lymphocyte (CTL) response. Depending on the immunization regimen used, Sendai virus-specific CTL responses were restricted to the H-2 antigens of either the stem cell donor or the thymus. Similarly, unprimed splenic T cells of normal mice were found to contain CTL-precursor cells that specifically reacted against Sendai virus or trinitrophenyl derivatives in the context of allogeneic major histocompatibility complex determinants that had not been encountered during their thymic differentiation. A frequency analysis of allogeneically versus syngeneically restricted virus-specific CTL precursors present in splenic T cells showed a ratio of about 1 to 6. These results provide evidence that H-2 restriction of trinitrophenyl- or Sendai virus-specific T cells is dictated by the complex type of the antigen-presenting cell and thus appears to be independent of the type of thymus in which the T cells have undergone maturation.
The structural polypeptides of aphthovirus are phosphoproteins.
Authors: La Torre J L , Grubman M J , Baxt B , Bachrach H L
Published: May 2006

Analysis of aphthovirus A12, strain 119ab, grown in the presence of inorganic 32P revealed that two of the major viral polypeptides, VP4 and trypsin-sensitive protein VP3, were highly phosphorylated. The other major polypeptides, VP1 and VP2, were also phosphorylated but to a much lesser extent. Polypeptides VP0 and P56, of which there are approximately one of two copies per aphthovirion, were also labeled with 32P. Phosphoserine and phosphothreonine appeared to be the amino acids labeled with 32P.
A species near its equilibrium size in a fluctuating environment can evolve a lower intrinsic rate of increase
Authors: Heckel David G. , Roughgarden Jonathan
Published: May 2006

In one specific model of a density-regulated population undergoing natural selection in a fluctuating environment there is a systematic evolutionary pressure favoring a lower intrinsic rate of increase, which can sometimes even overcome an evolutionary pressure favoring a higher carrying capacity.
A subpopulation of cultured human promyelocytic leukemia cells (HL-60) displays the formyl peptide chemotactic receptor.
Authors: Niedel J , Kahane I , Lachman L , Cuatrecasas P
Published: May 2006

The chemotactic peptide N-formyl-Nle-Leu-Phe-Nle-Tyr(125I)-Lys (formyl 125I-peptide) binds in a saturable manner to human promyelocytic leukemia cells (HL-60) grown in suspension culture. The binding is of high affinity (50% binding at 0.4--0.75 nM), is rapid (half-time at 22 degrees C = 10 min), is enhanced by divalent cations, but is poorly reversible. These characteristics are similar to those of the formyl peptide chemotactic receptor on human peripheral blood neutrophils. The relative potencies of a series of synthetic peptides in inhibiting binding to the HL-60 cell receptor correlate closely with their potencies in inhibiting binding to the peripheral neutrophil receptor. However, whereas all peripheral human neutrophils display the receptor, only a subpopulation of the cultured cells will bind and internalize tetramethylrhodamine-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys. This subpopulation is distinct from the rest of the culture, displays morphological characteristics typical of well-differentiated promyelocytes, and appears to account for all of the measured fromyl 125I-peptide binding. Binding of both 125I-labeled and tetramethylrhodamine-labeled formyl peptide increases in response to treatment of the culture with polar compounds that induce cell differentiation. Cells from the differentiated culture demonstrate a chemotactic response to N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys, whereas cells from the undifferentiated culture do not.
Isolation and characterization of polyoma virus mutants able to develop in embryonal carcinoma cells.
Authors: Vasseur M , Kress C , Montreau N , Blangy D
Published: May 2006

Embryonal carcinoma (EC) mouse cells have been shown to be resistant to infection by retroviruses and small oncogenic DNA viruses, including simian virus 40 and polyoma. When allowed to differentiate, in vitro or in vivo, EC cells become as susceptible to these viruses as differentiated mouse cell lines are. In order to study the relationships between differentiation of EC cells and viral expression, we have isolated and characterized several polyoma mutants that can express early and late functions in undifferentiated EC cells. These mutants, which arose spontaneously during high-multiplicity infection of PCD3 cells (a differentiated fibroblast-like cell line derived from PCC3 EC cells), were selected on PCC4 cells (undifferentiated EC cells) and twice plaque purified. Restriction enzyme analysis of the DNA from several mutants has shown that they all exhibit an additional sequence located in the Pvu II endonuclease fragment 4, close to the junction between Hpa II endonuclease fragments 3 and 5. The size of the insertion varies from 10 to 50 base pairs. The biological properties, including oncogenicity, transforming ability, host range, and burst size of the mutants so far analyzed are similar to those of wild-type virus.
Cell surface polypeptides of murine T-cell clones expressing cytolytic or amplifier activity.
Authors: Sarmiento M , Glasebrook A L , Fitch F W
Published: May 2006

Murine cytolytic T-cell and amplifier T-cell clones derived from secondary unidirectional mixed leukocyte cultures were labeled with 125I by the lactoperoxidase method and their polypeptide profiles were analyzed by NaDodSO4/polyacrylamide gel electrophoresis. All cytolytic T-cell clones derived from the same mouse strain yeilded similar cell surface polypeptide profiles. However, profiles obtained with three amplifier T-cell clones were strikingly different from each other as well as from those of cytolytic T-cell clones. Comparison of these profiles with those obtained from mixed leukocyte culture cells, whole spleen cells, glass wool/nylon wool-purified splenic T-cells, and thymocytes indicates that cytolytic T-cell clones have a characteristic cell surface polypeptide profile that distinguishes this type of lymphocyte from most other lymphocytes.
Allogeneic hemopoietic stem cell transplantation using mouse spleen cells fractionated by lectins: in vitro study of cell fractions.
Authors: Reisner Y , Ikehara S , Hodes M Z , Good R A
Published: May 2006

Mouse spleen cells sequentially agglutinated by soybean agglutinin (SBA) and peanut agglutinin (PNA) previously had been shown to be sufficiently depleted of graft-versus-host activity to allow reconstitution of lethally irradiated allogeneic recipient mice. We have now tested the extent of T-cell depletion in this cell fraction by various in vitro assays, including cytotoxicity testing with anti-Thy-1 antiserum, mitogenic response to the T-cell mitogens concanavalin A and phytohemagglutinin and allogeneic responsiveness in the mixed lymphocytes culture assay. By these criteria the SBA+, PNA+ spleen fraction, used previously in the in vivo experiments, was found to possess about 1% T-cell contamination. The slight contamination with T cells previously found in the singly agglutinated SBA fraction can be removed by a second fractionation with SBA, thus eliminating the possibility that a minor T-cell subpopulation bears receptors for SBA. Finally, we demonstrated that the twice-agglutinated fraction, by SBA and PNA or by SBA alone, contains a significant number of prothymocytes, thereby indicating that mouse prothymocytes bear receptors for both SBA and PNA. The implication of these findings to bone marrow transplantation in humans is discussed.
Phencyclidine interactions with the ionic channel of the acetylcholine receptor and electrogenic membrane
Authors: Albuquerque Edson X. , Tsai Ming-Cheng , Aronstam Robert S. , Witkop Bernhard , Eldefrawi Amira T. , Eldefrawi Mohyee E.
Published: May 2006

The effects of phencyclidine (PCP) were studied on the electrogenic and chemosensitive properties of the neuromuscular junction of skeletal muscle as well as on the binding sites on the acetylcholine (AcCho) receptor and its ionic channel in the electric organ membranes of the electric ray. The directly elicited muscle twitch was markedly potentiated by prolonging the falling phase of the muscle action potential and blocking delayed rectification. The indirectly elicited muscle twitch was transiently potentiated and then blocked by PCP at concentrations below 60 μM. PCP blocked miniature endplate potentials and AcCho sensitivities at the junctional region of innervated muscle, blocked the extrajunctional sensitivity of the chronically denervated muscle, and significantly depressed the peak amplitude of the endplate current (EPC) in a voltage- and time-dependent manner. PCP also caused acceleration of the time course of EPC decay and shortening of the mean life-time of the open ionic channel. The effects of PCP were not due to inhibition of AcCho receptor sites because PCP did not protect against the quasi-irreversible inhibition of receptor sites by α-bungarotoxin, nor did it inhibit binding of [
3
H]AcCho or [
125
I-labeled α-bungarotoxin to the receptor sites. On the other hand, PCP blocked the binding of [
3
H]perhydrohistrionicotoxin to the sites of the ionic channel of the AcCho receptor. The data suggest that PCP reacts with the electrogenic K
+
channel and the ionic channel associated with the AcCho receptor in the open as well as the closed conformation.
Anticholinergic substances: A single consistent conformation
Authors: Pauling Peter , Datta Narayandas
Published: May 2006

An interactive computer-graphics analysis of 24 antagonists of acetylcholine at peripheral autonomic post-ganglionic (muscarinic) nervous junctions and at similar junctions in the central nervous system, the crystal structures of which are known, has led to the determination of a single, consistent, energetically favorable conformation for all 24 substances, although their observed crystal structure conformations vary widely. The absolute configuration and the single, consistent (ideal) conformation of the chemical groups required for maximum anticholinergic activity are described quantitatively.
Corrected splicing of a chicken ovalbumin gene transcript in mouse L cells.
Authors: Breathnach R , Mantei N , Chambon P
Published: May 2006

Mouse thymidine-kinase-negative L cells were transformed with a cloned chicken ovalbumin gene linked to the cloned thymidine kinase gene from herpes simplex virus type 1. Most thymidine-kinase-positive clones contained one or more copies of the ovalbumin gene, which were stably maintained for at least 6 months during continuous culture under selective conditions. Transcription of the ovalbumin gene was detected at a low level, producing RNA molecules that were correctly spliced and polyadenylylated by comparison with genuine ovalbumin mRNA. However, the 5' end of these RNA molecules does not correspond to that of ovalbumin mRNA.
Coated vesicles transport newly synthesized membrane glycoproteins from endoplasmic reticulum to plasma membrane in two successive stages.
Authors: Rothman J E , Fine R E
Published: May 2006

The G protein of vesicular stomatitis virus is a transmembrane glycoprotein that is transported from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane via the Golgi apparatus. Clathrin-coated vesicles have been purified from CHO cells infected with vesicular stomatitis virus and shown to contain G protein in amounts nearly stoichiometric with clathrin. Pulse-chase experiments have demonstrated that this G protein is a transit form and have revealed that G is transported to the cell surface in two successive waves of coated vesicles. The oligosaccharides of G1 protein carried in the early wave are of the "high-mannose" variety which can be cleaved by the enzyme endoglycosidase H; the oligosaccharides of G2 protein in the second, later wave are resistant to endoglycosidase H. The early wave is therefore proposed to correspond to transport of G protein in coated vesicles from the endoplasmic reticulum to the Golgi apparatus, where the oligosaccharides are processed and resistance to endoglycosidase H is conferred; the succeeding wave would represent transport from the Golgi apparatus to the plasma membrane.
Properties of an acid phosphatase in pulmonary surfactant.
Authors: Benson B J
Published: May 2006

Lung surfactant, a lipid-protein complex purified from dog lungs, contains a highly active phosphomonoesterase associated with it. This phosphatase is quite specific for the hydrolysis of phosphatidic acid and 1-acyl-2-lysophosphatidic acid. The enzyme possesses many of the characteristics of the microsomal enzyme, phosphatidate phosphohydrolase (EC 3.1.3.4). In addition, we have shown that this enzyme will also convert phosphatidylglycerol phosphate [1-(3-sn-phosphatidyl)-sn-glycerol-1-P] to phosphatidylglycerol [1-(3-sn-phosphatidyl)-sn-glycerol] and Pi. The phosphatidylglycerol phosphate was made available to the surfactant enzyme in a coupled assay by hydrolysis of cardiolipin [1-(3-sn-phosphatidyl)-3-(3-sn-phosphatidyl)-sn-glycerol] by stereospecific cleavage with phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Bacillus cereus. This enzyme has been previously shown to generate the naturally occurring isomer of phosphatidylglycerol phosphate because it has specificity for the 3-(3-sn-phosphatidyl) group of cardiolipin. Other properties of the surfactant enzyme are discussed in relation to its presence in lung surface active material.
Protein phosphorylation and sodium conductance in nerve membrane.
Authors: Schoffeniels E , Dandrifosse G
Published: May 2006

High molecular weight proteins extracted from the walking nerves of the shore crab (Carcinus maenas) exhibit a cycle of phosphorylation-dephosphorylation that is influenced by neurotropic compounds and inorganic ions. The net phosphorylation state of the proteins is increased in the presence of K+ ions and decreased with Na+ ions. In the absence of Mg2+ there is no phosphorylation. Ca2+ ions at low concentrations are necessary for optimal phosphorylation. At high concentration (above 0.1 mM), Ca2+ ions are inhibitory. Neurotropic compounds generally inhibit the phosphorylation process. More specifically, tetrodotoxin and veratridine, depending on the ionic composition of the medium, have opposite effects on the phosphorylation process, a result in agreement with their known physiological action. It is suggested that the high molecular weight components thus identified are part of the sodium permeation sites and that the conductance state of those sites is controlled by a phosphorylation process.
Characterization of double-stranded-RNA-activated kinase that phosphorylates α subunit of eukaryotic initiation factor 2 (eIF-2α) in reticulocyte lysates
Authors: Levin Daniel H. , Petryshyn Ray , London Irving M.
Published: May 2006

Incubation of reticulocyte lysates with low levels of double-stranded (ds) RNA (1-20 ng/ml) activates a cAMP-independent protein kinase (dsI) that phosphorylates the α-subunit (
M
r
38,000) of initiation factor 2 (eIF-2) and produces an inhibition of protein chain initiation similar to that caused by heme deficiency. Activation of dsI from its latent precursor takes place on the ribosomes and requires ATP. dsI can also be activated in ribosomal salt washes and in partially purified preparations of the latent precursor of dsI. In all preparations, activation is accompanied by the ds RNA-dependent phosphorylation of a polypeptide doublet that migrates as bands of 67 and 68.5 kilodaltons (67/68.5) in NaDodSO
4
/acrylamide gels. The rate of phosphorylation of these components in a ribosome salt wash is more rapid than the ds RNA-dependent phosphorylation of eIF-2α. Other polypeptides in the salt wash also undergo ds RNA-dependent phosphorylation, but their significance is not clear. All of these phosphorylations are prevented by high concentrations of poly(I)·poly(C)(20 μg/ml), but not by an antiserum specific for the heme-regulated eIF-2α kinase. Both the latent and activated forms of dsI have been partially purified from a 0.5 M KCl wash of reticulocyte ribosomes. The two species have similar
M
r
s (≈120,000) and sedimentation coefficients (≈3.75 S), which suggests that activation of dsI probably does not involve extensive changes. By comparison, the heme-regulated eIF-2α kinase has an
M
r
of ≈160,000 and sediments at ≈6.6 S. However,
in vitro
, dsI and HRI both phosphorylate the same site(s) of eIF-2α. Purified dsI inhibits protein synthesis in hemin-supplemented lysates with the same kinetics induced by the addition of ds RNA; both inhibitions are reversed by eIF-2. dsI that has been activated in the salt wash and then purified does not require ds RNA for expression and no longer displays phosphorylation of the 68.5/67 doublet, which appears to occur only during activation. The data support the view that this component(s) may be the eIF-2α kinase activated by ds RNA.