bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutants altering coordinate synthesis of specific myosins during nematode muscle development.
Authors: Zengel J M , Epstein H F
Published: May 2006

Mutations in the unc-52 gene on linkage group II retard the construction of body-wall muscle sarcomeres during larval development in the nematode Caenorhabditis elegans. Unc-52 mutants show decreased accumulation of myosin heavy chains relative to other polypeptides during larval development, correlating with the structural retardation. Pulse radiolabeling experiments show that decreased synthesis of specific body-wall myosin heavy chains that are encoded by the unc-54 gene on linkage group I is responsible for the defective myosin accumulation. In the wild type, a constant ratio of the synthesis of the unc-54-coded myosin B to myosin A, about 2:1, is maintained during the larval stages in which the synthesis of both myosins increases exponentially and rapid sarcomere growth and addition ensues. During the first 26 hr of larval development, before any structural or behavioral effects of unc-52 mutations are apparent, the synthesis of myosin heavy chains is also normal. By 38 hr, decreased synthesis of myosin B is detected in the unc-52 mutant SU200, when sarcomere growth slows considerably. The effects of mutation in the unc-52 locus are trans acting upon the synthesis of unc-54-coded myosin in a specific set of muscle cells during a defined period of larval development.
Construction of recombinant plasmids containing rat muscle actin and myosin light chain DNA sequences.
Authors: Katcoff D , Nudel U , Zevin-Sonkin D , Carmon Y , Shani M , Lehrach H , Frischauf A M , Yaffe D
Published: May 2006

The construction and partial characterization of recombinant bacterial plasmids carrying DNA sequences that hybridize with rat skeletal muscle actin and a myosin light chain mRNA is described. DNA of one clone hybridizes specifically with the muscle-specific alpha-actin mRNA. Three plasmid clones contain DNA inserts that hybridize with muscle as well as with nonmuscle actin mRNA. A fifth plasmid contains sequences complementary to mRNA coding for myosin light chain 2. DNA of this plasmid hybridizes specifically with RNA extracted from muscle and differentiated muscle cultures but not with RNA extracted from proliferating mononucleated myogenic cells.
Electron paramagnetic resonance crystallography of
17
O-enriched oxycobaltomyoglobin: Stereoelectronic structure of the cobalt dioxygen system
Authors: Dickinson L. Charles , Chien James C. W.
Published: May 2006

An electron paramagnetic resonance crystallographic study was made on oxycobaltomyoglobin with the dioxygen ligand enriched to 19.1% in
17
O. There are two spectroscopically distinct cobalt dioxygen species. The less abundant species, II (40%), has nonequivalent oxygen atoms with superhyperfine tensors
O
A
α
= (5, -67.5, 22.4)G and
O
A
β
= (5.4, -83.3, 30.3)G. Together with the previously reported
59
Co hyperfine tensor [Chien, J. C. W. & Dickinson, L. C. (1972)
Proc. Natl. Acad. Sci. USA
69, 2783-2787], the orbital spin densities are found to be O
α
(
p
η
) = 0.48, O
α
(
p
ζ
) = -0.11, O
β
(
p
η
) = 0.74, O
β
(
p
ζ
) = -0.16, Co(

d
xz

) = -0.01, Co(

d
yz

) = 0.06 for a total electron density of 1.01. The O—O axis is directed toward His-E7, suggesting a possible hydrogen bonding interaction which may contribute to the nonequivalency of the oxygen atoms; its projection approximately bisects N
1
—Fe—N
2
. The
z
axis of the
Co
A tensor is tilted at an angle of 28° from the heme normal, resulting in a Co—O—O angle of 120°. The more abundant species, I (60%), has equivalent oxygen atoms with
O
A
γ
= (12, -72.5, 20)G and orbital spin densities of O
γ
(
p
η
) = 0.54, O
γ
(
p
ζ
) = -0.05, Co(

d
xz

) = -0.02, Co(

d
yz

) = 0.09 for a total spin density of 1.10. Although the direction cosines for this molecule cannot be precisely determined, the projection of its O—O axis approximately bisects N
2
—Fe—N
3
and is parallel to the imidazole ring of His-F8. Increase of temperature changes g,
Co
A, and
O
A values, with the largest effect seen with
O
A. This temperature dependence indicates averaging of the two bond structures which are stabilized at 77 K.
Structural and functional analysis of cloned DNA containing genes responsible for branched-chain amino acid transport in Escherichia coli.
Authors: Oxender D L , Anderson J J , Daniels C J , Landick R , Gunsalus R P , Zurawski G , Selker E , Yanofsky C
Published: May 2006

The four genes encoding the components of the high-affinity branched-chain amino acid transport systems in Escherichia coli (livH, livG, livJ, and livK) have been cloned into lambda phage and subsequently into the plasmid vector pACYC184. The presence of the four structural genes and their accompanying regulatory regions on the resultant plasmid, pOXI, was confirmed by genetic complementation and analysis and by transport studies carried out on the appropriate transformed mutant strains. When pOX1 DNA was used to direct an in vitro transcription/translation system, four major polypeptide products were produced. Immunoprecipitation with antibody directed against the LIV-binding protein identified the two leucine-binding proteins as products of in vitro synthesis. The binding proteins were produced in precursor forms and had molecular weights approximately 2500 higher than the processed, mature forms. A minicell-producing strain transformed with plasmid pOX1 produced the binding proteins in the processed form.
Rearrangement of immunoglobulin heavy chain genes during B-lymphocyte development as revealed by studies of mouse plasmacytoma cells
Authors: Coleclough C. , Cooper D. , Perry R. P.
Published: May 2006

We have used Southern's blotting technique to determine the extent to which the genes encoding the constant (C) regions of μ, α, γ
1
, and γ
2b
immunoglobulin heavy (H) chains are altered in number and context from their germline (embryo) state in a series of 14 plasmacytomas expressing various H chain classes. In the three μ chain-producing plasmacytomas studied there was no evidence of rearrangement of C
H
genes other than C
μ
. In contrast, rearrangement and deletion of nonexpressed C
H
genes was frequent in plasmacytomas that produce γ or α chains. The observed pattern of deletions is consistent with the idea that the ontogenetic switch in H chain class requires C
H
gene deletion. Frequently, though not always, such deletions as well as other types of rearrangement occur in both allelic loci. Particularly noteworthy are three γ
2a
-expressing tumors in which C
α
gene rearrangement is evident in both alleles. We incorporate these observations into a probabilistic model of B cell development: in the first phase, deletions may occur between the C
μ
gene and the variable (V
H
) gene array, which result in the formation of a productive fused V
H
-C
μ
gene. The cell may then enter a second phase, which allows deletions within the C
H
gene arrays of both homologous chromosomes. Some deletions juxtapose the expressed V
H
gene with a second C
H
gene and result in a H chain class switch; others delete or alter the context of C
H
genes without changing the phenotype of the cell. We predict that switching can be both a single-step and a multi-step process, and that in the latter case those rearrangements that do not result in a switch may be physiologically significant in that they may limit the options of further switching.
Simian virus 40 and polyoma virus stimulate overall cellular RNA and protein synthesis.
Authors: Khandjian E W , Matter J M , Léonard N , Weil R
Published: May 2006

In lytic infection with simian virus 40 and polyoma virus of monkey and mouse cells in tissue culture, synthesis of the viral tumor (T) antigens (T antigens) is rapidly followed by a mitogenic response of the host cell. The latter begins with virus-induced stimulation of overall cellular RNA and protein synthesis, leading to a substantial increase in cytoplasmic and nuclear RNA and protein. Stimulation begins within 1 hr after onset of T-antigen synthesis and also occurs if virus-induced DNA synthesis is blocked by metabolic inhibitors. The broad spectrum of biological and molecular effects induced by simian virus 40 and polyoma virus is, at least phenotypically, reminescent of the pleiotropic impact exerted on target cells by nonviral mitogens and by certain growth-promoting steroid and polypeptide hormones.
Temperature-sensitive tumorigenicity of cells transformed by a mutant of Moloney sarcoma virus.
Authors: Klarlund J K , Forchhammer J
Published: May 2006

Normal rat kidney cells were nonproductively infected either with CP27, a mutant of Moloney sarcoma virus that is temperature-sensitive for maintenance of transformation, or with the parental wild-type virus. The nonproducer cells were inoculated into the tails of athymic nude mice that were subsequently incubated at 28 or 36 degrees C. CP27-infected cells induced tumors only at 28 degrees C, whereas cells infected with wild-type Moloney sarcoma virus were tumorigenic at both temperatures. Tumors induced at 28 degrees C by wild-type virus-infected cells grew faster after shift of the mice to 36 degrees C. In contrast, tumors induced by CP27-infected cells regressed upon shift to 36 degrees C, indicating that continuous expression of viral functions is required for persistence and growth of the tumors. After regression, secondary tumor growth was observed late after upshift of temperature-sensitive tumors. Cells recovered from these late-appearing tumors were tumorigenic at the nonpermissive temperature, and tumors induced by these cells did not regress after upshift. Virus rescued from these recovered cells retained the temperature-sensitivity for focus formation, indicating that the occurrence of the phenotypically wild-type cells was due to host cell modifications rather than to reversion of the CP27 genome.
Control of differentiation of a mammary cell line by lipids.
Authors: Dulbecco R , Bologna M , Unger M
Published: May 2006

A rat mammary cell line (LA7) undergoes spontaneous differentiation into domes due to production of specific inducers by the cells. Some of these inducers may be lipids, and we show that lipids regulate this differentiation as both inducers and inhibitors. One inhibitor is the tumor promoter tetradecanoyl-13 phorbol 12-acetate. The inducers are saturated fatty acids of two groups: butyric acid and acids with chain lengths from C13 to C16, especially myristic acid (C14). Other inducers are myristoyl and palmitoyl lysolecithins, myristic acid methyl ester, and two cationic detergents with a tetradecenyl chain. We propose that the lipids with a C14-C16 alkyl chain affect differentiation by recognition specific receptors through their alkyl chains and that the effects obtained depend on the head groups. These lipids may be physiological regulators in the mammary gland.
Proteins iodinated by the Chloramine-T method appear to be degraded at an abnormally rapid rate after endocytosis
Authors: Opresko L. , Wiley H. S. , Wallace R. A.
Published: May 2006

Proteins labeled with either
3
H by reductive methylation or
125
I by the chloramine-T method were incubated with
Xenopus laevis
oocytes; the incorporation and acid precipitability of the proteins were then studied. The uptake rates of both specifically incorporated (vitellogenin) and nonspecifically incorporated proteins (bovine serum albumin and
X. laevis
serum proteins lacking albumin) were not influenced by the method of labeling. However,
125
I-labeled proteins were apparently degraded at rates far exceeding their
3
H-labeled counterparts, based on the generation of acid-soluble radioactivity. Thus, after a 3-hr incubation, 3-5 times more
125
I-labeled bovine serum albumin and
X. laevis
serum proteins lacking albumin were degraded than the corresponding
3
H-labeled proteins (95% compared to 30% and 75% compared to 15%, respectively), whereas after a 24-hr incubation, the degradation of
125
I-labeled vitellogenin was 15 times greater than that of [
3
H]vitellogenin labeled
in vivo
(60% compared to 4%). Moreover, examination of the relative amounts of
3
H- compared to
125
I-labeled bovine serum albumin deposited into the exogenously derived yolk platelet compartment of the oocyte revealed 7 times more acid-precipitable
3
H-labeled protein, indicating that the observed discrepancies were not due to reincorporation of the
3
H-labeled ligands. Passage of dissolved oocytes previously exposed to
125
I-labeled bovine serum albumin (chloramine-T method) over a column of Bio-Gel P-10 revealed some breakdown of bovine serum albumin to intermediate molecular weight components and the presence of a large amount (≈90%) of labeled low molecular weight compounds, which analysis showed to be 72% free iodine. The evolution of either iodotyrosine or free iodine would nevertheless be perceived as protein degradation by most analytical procedures (e.g., acid precipitation or autoradiography). We conclude, therefore, that apparent degradation rates observed for endocytotically incorporated proteins may vary depending on the method used to label the protein and caution should be exercised when interpreting results obtained with labeled, particularly chloramine-T labeled, proteins.
Binding of sea anemone toxin to receptor sites associated with gating system of sodium channel in synaptic nerve endings in vitro.
Authors: Vincent J P , Balerna M , Barhanin J , Fosset M , Lazdunski M
Published: May 2006

Iodination of toxin II from the sea anemone Anemonia sulcata gives a labeled monoiododerivative that retains 80% of the original neurotoxicity. This derivative binds specifically to rat brain synaptosomes at 20 degrees C and pH 7.4 with a second-order rate constant of association ka = 4.6 x 10(4) M-1 sec-1 and a first-order rate constant of dissociation kd = 1.1 x 10(-2) sec-1. The binding occurs on the Na+ channel at a binding site distinct from that of other gating system toxins like batrachotoxin, veratridine, grayanotoxin, aconitine, and pyrethroids. The maximal binding capacity Bmax is 3.2 pmol/mg of protein (i.e., about two sea anemone toxin binding sites per tetrodotoxin binding site) and the Kd is 240 nM for the monoiododerivative and 150 nM for the native toxin. Corresponding binding parameters for the association of a 125I-labeled derivative of toxin II from the scorpion Androctonus australis Hector are Bmax = 0.3 pmol/mg of protein and Kd = 1 nM, whereas the Kd of the unmodified scorpion toxin is 0.6 nM. Competition experiments involving scorpion toxins, sea anemone toxins, and synaptosomes demonstrate that, although the sea anemone toxin is able to displace the scorpion toxin bound to synaptosomes, the scorpion toxin does not displace the sea anemone toxin. The sea anemone toxin but not the scorpion toxin binds to depolarized synaptosomes. Differences between binding properties of the two polypeptide toxins are analyzed in the discussion.
Intracellular distribution of nerve growth factor in rat pheochromocytoma PC12 cells: evidence for a perinuclear and intranuclear location.
Authors: Marchisio P C , Naldini L , Calissano P
Published: May 2006

The distribution of nerve growth factor (NGF) in rat pheochromocytoma cells (clone PC12) has been studied with two different techniques: immunofluorescence and autoradiography. It was found that NGF is progressively internalized in the cytoplasmic compartment and eventually accumulates in the form of discrete dots around the nucleus. A fraction of the internalized NGF appears within the nucleoplasm, often contiguous with the nucleolus. It is suggested that cytoplasmic and perinuclear NGF may be be in contact with a pool of tubulin or actin-like proteins in their soluble or organized form and play a key role in the process of arrest of division and neurite growth.
Interaction between cytochrome b5 and hemoglobin: involvement of beta 66 (E10) and beta 95 (FG2) lysyl residues of hemoglobin.
Authors: Gacon G , Lostanlen D , Labie D , Kaplan J C
Published: May 2006

In erythrocytes the reduction of oxidized hemoglobin (methemoglobin) is dependent upon an electron transport reaction between cytochrome b5 and methemoglobin. These two proteins are believed to form a complex whose bonding is principally determined by complementary charge interactions between acidic groups of cytochrome b5 and basic groups of hemoglobin. In order to refine this model, three surface lysyl hemoglobin variants--namely Hb N Baltimore beta 95 (FG2) Lys leads to Glu, Hb I Toulouse beta 66 (E10) Lys leads to Glu, and Hb I Philadelphia alpha 16 (A14) Lys leads to Glu--have been studied with respect to their reducibility and ability to bind cytochrome b5. In the two former variants, the substituted amino acids are located near the heme crevice; in the third one the substitution lies far from it. Substitutions of lysine for glutamic acid in positions beta 66 and beta 95 perturb the formation of the cytochrome b5--hemoglobin complex and result in a dramatic impairment of the cytochrome b5-mediated reduction, whereas the same mutation in position alpha 16 has no effect. We conclude that the lysine residues in positions beta 66 and beta 95 are directly involved in the binding of cytochrome b5. The three-dimensional structure of hemoglobin suggests that the cytochrome b5-binding domain of hemoglobin is constituted by four lysine residues surrounding the heme crevice in both alpha and beta chains. Similarities with other interacting hemoproteins are discussed.
In vitro
synthesis of pre-proteins of vacuolar compartmented proteinase inhibitors that accumulate in leaves of wounded tomato plants
Authors: Nelson Christopher E. , Ryan Clarence A.
Published: May 2006

Two proteinase inhibitor proteins that are compartmented in leaf vacuoles (lysosomes) were synthesized
in vitro.
mRNA was isolated from 17-day-old expanding tomato leaves by extraction with chaotropic buffers followed by chromatography on oligo(dT)-cellulose and was translated with a rabbit reticulocyte lysate system. Preparations of mRNA from leaves of both wounded plants and unwounded plants directed the incorporation of equivalent amounts of label into trichloroacetic acid-precipitable proteins. Only mRNA from leaves of wounded plants directed label into proteins that could be immunoprecipitated with rabbit IgG specific for either inhibitor I or inhibitor II. These results indicate that the wound-induced accumulation of proteinase inhibitors I and II in leaf vacuoles is a result of the presence of translatable mRNA species not present in leaves of unwounded plants. Gel electrophoresis of the immunoprecipitates in NaDodSO
4
/urea/polyacrylamide gels revealed that inhibitors I and II were translated
in vitro
as precursors about 2000 daltons larger than the inhibitors found in leaves. The presence of the additional polypeptide sequences in the newly synthesized inhibitors indicates that the inhibitors are processed either during or after synthesis, and the presequences may be signal peptides that are part of the process of inhibitor transport into the vacuolar compartments of tomato leaf cells.
Immunological differences between actins from cardiac muscle, skeletal muscle, and brain
Authors: Morgan Janet L. , Holladay Carter R. , Spooner Brian S.
Published: May 2006

The antigenic similarities and differences between various actins were explored by use of antisera against purified bovine cardiac actin and chicken embryo brain actin. In double-antibody coprecipitation tests, purified iodinated actins from bovine cardiac muscle, rabbit skeletal muscle, chicken embryo brain, and bovine brain all bound to antiserum against chicken embryo brain actin. This result demonstrates the presence of shared antigenic determinants among these actins. Cardiac actin antiserum, on the other hand, bound cardiac and skeletal actin, but failed to bind significantly either brain actin. In radioimmunoassay, all four unlabeled actins were capable of some degree of inhibition of binding of
125
I-labeled chicken embryo brain actin to homologous antiserum. The results confirm the existence of shared or similar antigenic determinants, but also show that the molecules are not antigenically identical. In the cardiac actin radioimmunoassay, unlabeled cardiac and skeletal muscle actins inhibited the binding of
125
I-labeled cardiac actin to homologous antiserum, but neither brain actin inhibited the binding. Thus, the muscle actins possess at least one antigenic determinant not expressed by the brain actins, in addition to the shared determinants. Furthermore, cardiac actin and skeletal actin generated different inhibition curves in the cardiac actin radioimmunoassay, demonstrating that, although antigenically related, they are not identical. Correlations with existing sequence data imply that substitutions in only a few residues alter the antigenic properties of actin.
Cyclic AMP as a negative regulator of hormonally induced lactogenesis in mouse mammary gland organ culture.
Authors: Perry J W , Oka T
Published: May 2006

In organ cultures of mammary glands from mice in midpregnancy, addition of both insulin and prolactin induces a marked accumulation of alpha-lactalbumin, whereas the augmentation of casein synthesis requires the presence of insulin, prolactin, and cortisol. Addition of 0.5 mM dibutyryl cyclic AMP resulted in complete inhibition of alpha-lactalbumin accumulation and partial inhibition of casein synthesis. Furthermore, either cholera toxin at 0.1-1.0 microgram/ml (a stimulator of adenylate cyclase) or 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase) in combination with 2 mM cyclic AMP, produced a similar pattern of inhibition of alpha-lactalbumin and casein synthesis in cultured tissue. During culture of mammary explants in medium containing no hormone, or insulin alone, or insulin, prolactin, and cortisol, the tissue content of cyclic AMP decreased rapidly, reaching half the initial level in 24-48 hr. These results indicate that cyclic AMP plays "negative" regulatory function in hormonal induction of milk protein synthesis during the development of the mammary gland.
Nucleotide sequence divergence of mouse immunoglobulin gamma 1 and gamma 2b chain genes and the hypothesis of intervening sequence-mediated domain transfer.
Authors: Miyata T , Yasunaga T , Yamawaki-Kataoka Y , Obata M , Honjo T
Published: May 2006

The nucleotide sequences of the constant portions of mouse immunoglobulin gamma 1 and gamma 2b genes were compared. A remarkable homology was found in a long (about 500 nucleotides) continuous segment including the entire CH1 coding region and about the first half of the first intervening sequence. Furthermore, comparison of amino acid sequences of four gamma-class chains revealed that the CH1 domain shows limited divergence among gamma 1, gamma 2a, and gamma 2b. Interestingly, the homology region extends to the CH2 domain in gamma 2a and gamma 2b. These findings suggest that, during their evolution, a double unequal crossing-over event has taken place at different intervening sequences, resulting in the transfer of the DNA segment coding for the CH1 domain or CH1-CH2 domains. A possible evolutionary implication for such an "intervening sequence-mediated domain transfer" event is discussed.
Protease-activated “prodrugs” for cancer chemotherapy
Authors: Carl Philip L. , Chakravarty Prasun K. , Katzenellenbogen John A. , Weber Michael J.
Published: May 2006

Many types of malignant cells and human tumors display increased concentrations of the protease plasminogen activator that converts plasminogen to the highly active protease, plasmin. Because plasmin rapidly cleaves various low molecular weight compounds coupled to appropriate peptide specifiers, we hypothesized that coupling of such peptide specifiers to anticancer drugs might create “prodrugs” which would be locally activated by tumor-associated plasmin and consequently would be less toxic to normal cells. To provide an initial test of this concept we have synthesized peptidyl prodrugs of the structure D-Val-Leu-Lys-X in which the peptidyl portion has been designed to allow the prodrug to serve as an excellent plasmin substrate and X is an anticancer drug—either the glutamine analog (α
S
,5
S
) α-amino-3-chloro-4,5-dihydro-5-isoxazole-acetic acid (AT-125) or the alkylating agent
N,N
-bis(2-chloroethyl)-
p
-phenylenediamine (phenylenediamine mustard). Treatment of these prodrugs with plasmin generated the free peptide and the free drug, demonstrating that these prodrugs are plasmin substrates. The prodrugs and free drugs were tested in an
in vitro
system against either normal chicken embryo fibroblasts, which display a low level of plasminogen activator, or their virally transformed counterparts, which produce high levels of plasminogen activator. In each case the peptidyl prodrugs displayed at least a 5-fold increase in selectivity for the transformed cells compared to the free drug. The greater selectivity of action of the peptidyl prodrugs against transformed cell cultures suggests that these or similar prodrugs that are substrates for tumor-associated proteases may show increased therapeutic effectiveness in the treatment of tumors that produce sufficiently increased amounts of plasminogen activator.
Adrenocortical response to corticotropin is potentiated by part of the amino-terminal region of pro-corticotropin/endorphin.
Authors: Pedersen R C , Brownie A C
Published: May 2006

Five peptides derived from pro-corticotropin/endorphin (pro-ACTH/endorphin), the pituitary corticotroph cell prohormone, were bioassayed with isolated rat adrenocortical cells: alpha- and beta-melanotropin, beta-lipotropin, beta-endorphin, and the amino-terminal region of pro-ACTH/endorphin known as "16k fragment." The effect of each on steroidogenesis was measured at potentially physiological concentrations (0.01-1 nM) in both the absence and presence of varying concentrations of ACTH-(1-24). Of the peptides tested, only 16k fragment, the amino-terminal region of pro-ACTH/endorphin, has a slight but significant potentiating effect on ACTH-(1-24) action. Prior treatment of 16k fragment with trypsin for 30 sec dramatically increases this dose-dependent synergism. Experiments performed in vivo with hypophysectomized female rats indicate that the trypsin digest of 16k fragment stimulates cholesterol ester hydrolase (cholesterol esterase; sterol-ester acylhydrolase, EC 3.1.1.13) activity in the adrenal cortex but fails to activate cholesterol side-chain cleavage. The effect of the trypsinized material can therefore be qualitatively distinguished from that of ACTH-(1-24). When both ACTH-(1-24) and the digest are administered together, a synergistic increase in serum corticosterone concentration results. We propose that a portion of 16k fragment molecule may play a hormonal role in the control of adrenocortical steroidogenesis.
Urinary and brain beta-carboline-3-carboxylates as potent inhibitors of brain benzodiazepine receptors.
Authors: Braestrup C , Nielsen M , Olsen C E
Published: May 2006

Benzodiazepines probably exert their anxiolytic, hypnotic, and anticonvulsant effects by interacting with brain-specific high-affinity benzodiazepine receptors. In searching for possible endogenous ligands for these receptors we have purified a compound 10(7)-fold from human urine by extractions, treatment with hot ethanol, and column chromatography. The compound was identified as beta-carboline-3-carboxylic acid ethyl ester (IIc) by mass spectrometry, NMR spectrometry, and synthesis; IIc was also isolated from brain tissues (20 ng/g) by similar procedures. Very small concentrations of IIc displaced [3H]diazepam completely from specific cerebral receptors, but not from liver and kidney binding sites; the concentration causing 50% inhibition of specific [3H]diazepam binding (IC50) was 4-7 nM compared to ca. 5 nM for the potent benzodiazepine lorazepam. Specific binding sites for quinuclidinyl benzilate, naloxone, spiroperidol, serotonin, muscimol, and WB 4101 were not affected by IIc. In contrast to benzodiazepines, IIc exhibits "mixed type" competitive inhibition of forebrain benzodiazepine receptors (negative cooperativity). We surmise that an endogenous ligand for benzodiazepine receptors may be a derivative of beta-carboline-3-carboxylic acid.
Right and left eye bands in frogs with unilateral tectal ablations.
Authors: Law M I , Constantine-Paton M
Published: May 2006

Surgical ablation of a single tectal lobe in Rana pipiens can cause regenerating retinal ganglion cell axons to cross to the remaining tectum. These synaptically deprived fibers can obtain termination space in a retinotopic and highly stereotyped manner. Each of the two eyes can share the undisturbed tectum by terminating in mutually exclusive, eye-specific stripes that alternate across the medial-lateral extent of the tectal lobe. Invading axons from the ipsilateral eye must actively displace established synapses from the contralateral eye in order to form these exclusive termination zones because the normal projection to the intact tectum is not severed in these experiments. In animals in which a large proportion of anomalous fibers do not reach the undisturbed tectum, only a few ipsilateral eye bands are observed. Nevertheless, these bands have the same width, periodicity, and orientation as those observed in fully banded preparations. When ipsilateral eye terminal density is extremely low, banding is absent. The completely striped termination pattern of unitectal animals is identical to the pattern previously reported in the dually innervated tecta of three-eyed R. pipiens. We theorize that this pattern results from a compromise between two synaptogenic forces that are active in regeneration as well as in development.
Circadian rhythms of melatonin release from individual superfused chicken pineal glands in vitro.
Authors: Takahashi J S , Hamm H , Menaker M
Published: May 2006

The pineal gland of birds contains one or more circadian oscillators that play a major role in overall temporal organization. We have developed a flow-through culture system for the isolated pineal by which we can measure the release of melatonin continuously from superfused glands over long periods of time. Chicken pineals release melatonin rhythmically, and these rhythms persist in vitro with a circadian oscillation. In light cycles the release of melatonin is strongly rhythmic; however, in constant conditions the amplitude of the rhythm is lower and appears to be damping. Light has at least two effects upon the isolated pineal: cyclic light input synchronizes the rhythm, and acute light exposure at night rapidly inhibits melatonin release. The cultured avian pineal clearly offers great potential as a model system for the study of vertebrate circadian oscillators and may open the way for an analysis of mechanism.
Locomotor activation induced by infusion of endorphins into the ventral tegmental area: evidence for opiate-dopamine interactions.
Authors: Stinus L , Koob G F , Ling N , Bloom F E , Le Moal M
Published: May 2006

beta-Endorphin in nanomole quantities produced a stimulation of locomotor activity when infused into the region of the dopamine cell bodies of the ventral tegmental area (VTA) in rats. alpha-, gamma-, and des-Tyr-gamma-endorphin produced similar effects, but the D-alanine analogues of alpha and gamma-endorphin produced a larger and longer-lasting activation, presumably reflecting their resistance to degradation. This locomotor activation was reversible by pretreatment with naloxone and by destruction of the terminal projections of the mesocorticolimbic dopamine system originating in the VTA. These results demonstrate that locally infused endorphin can interact with the opioid receptors in the VTA, and they suggest a means by which endorphins activate limbic excitability.
Purification and primary structure of two neuroactive peptides that cause bag cell afterdischarge and egg-laying in
Aplysia
Authors: Heller E. , Kaczmarek L. K. , Hunkapiller M. W. , Hood L. E. , Strumwasser F.
Published: May 2006

Two neuroactive peptides, A and B, have been isolated from the atrial gland in the reproductive tract of
Aplysia
. Each of the two peptides is able to induce egg-laying behavior in recipient animals.
In vitro
recordings from the abdominal ganglion show that both peptides also trigger longlasting discharges in the bag cell neurons at concentrations around 0.1 μM. The peptides were purified by a combination of ammonium sulfate precipitation, agarose gel filtration, and cation exchange chromatography. Each peptide has 34 amino acid residues. Microsequencing together with carboxypeptidase Y degradation and analysis of tryptic peptides revealed the following sequence for peptide A: H-Ala-Val-Lys-Leu-Ser-Ser-Asp-Gly-Asn-Tyr-Pro-Phe-Asp-Leu-Ser-Lys-Glu-Asp-Gly -Ala-Gln-Pro-Tyr-Phe-Met-Thr-Pro-Arg-Leu-Arg-Phe-Tyr-Pro-Ile. Peptide B differs from A in only four positions. The first nine residues of B are: Ala-Val-Lys-Ser-Ser-Ser-Tyr-Glu-Lys-, whereas residues 10-34 of B are identical to those of A. The calculated
M
r
of A is 3924 and that of B is 4032. The pI of peptide A as determined by isoelectric focusing in polyacrylamide gels is 7.9-8.1 and that of peptide B is 9.0-9.2. It is estimated that each atrial gland contains at least 150 μg of peptide A and 50 μg of B. Neither peptide resembles the egg-laying hormone isolated from bag cell neurons. It is postulated that the atrial gland peptides are released during copulation, and then by interacting with neuronal receptors in the head ganglia and pleuroabdominal connectives they cause the bag cells to afterdischarge, thereby releasing egg-laying hormone.
Luminescence of carbon monoxide hemocyanins.
Authors: Kuiper H A , Agrò A F , Antonini E , Brunori M
Published: May 2006

The effect of carbon monoxide on the luminescence properties of Helix pomatia alpha-hemocyanin and Panulirus interruptus hemocyanin has been studied. These proteins, when saturated with carbon monoxide, show, besides the intrinsic fluorescence arising from the aromatic amino acid residues, emission in the visible region with a maximum between 540 and 560 nm. Results of carbon monoxide titration experiments and data from absorption and excitation-emission spectra provide convincing evidence that the observed emission originates from a fluorescent copper(I)-carbon monoxide complex, and this emission is interpreted as charge-transfer luminescence.
Specific RNA-cleaving activities from HeLa cells.
Authors: Ferrari S , Yehle C O , Robertson H D , Dickson E
Published: May 2006

Subcellular fractionation of HeLa cells was carried out under gentle conditions to isolate enzymes that cleave RNA precursors in a specific manner. Four separate activities--cleavage of HeLa cell heterogeneous nuclear RNA, the HeLa cell 45S rRNA precursor, RNA . DNA hybrids (RNase H), and the Escherichia coli tRNATyr precursor (RNase P)--were revealed by these studies. The specificity and limited nature of these cleavages suggest that they are due to eukaryotic RNA-processing enzymes. The virtual absence of random nucleases from these enzymes was demonstrated by their inability to cleave the 8000-base early mRNA precursor of bacteriophage T7, E. coli 30S rRNA precursor, or HeLa cytoplasmic poly(A)-containing RNA.
Mechanism of interferon action: Simian virus 40-specific early polypeptides synthesized in untreated and interferon-treated monkey kidney cells
Authors: Kingsman Susan M. , Smith Mark D. , Samuel Charles E.
Published: May 2006

The effect of interferon treatment on proteins synthesized in simian virus 40 (SV40)-infected cells in the presence of cytosine arabinoside was investigated. The following results were obtained: (
i
) In addition to previously described large tumor (T) antigen (94 kilodaltons) and small tumor (t) antigen (19 kilodaltons), a 62-kilodalton polypeptide was immunoprecipitated by SV40 anti-T antiserum from extracts of infected CV-1 and BSC-1 monkey kidney cells and transformed SV3T3 mouse cells. The 94-, 62-, and 19-kilodalton polypeptides were not precipitated with normal serum from extracts of infected cells, and they were not present in extracts of uninfected cells. (
ii
) The
de novo
synthesis of the 94-, 62-, and 19-kilodalton tumor antigens was inhibited in CV-1 and BSC-1 cells treated with interferon before infection; total cellular protein synthesis was not significantly affected by interferon treatment. The relative interferon sensitivity of the three polypeptides in lytically infected monkey cells was comparable; by contrast, interferon did not affect their synthesis in transformed mouse cells. (
iii
) The 62-kilodalton polypeptide was detected in monkey cells infected with the following strains of SV40: tsA30 at both 33°C and 41°C; wt 708, the parent of tsA30; dI 884; and wt 830, the parent of dI 884. The amount of the 62-kilodalton species relative to T antigen was significantly greater in tsA30-infected cells as compared to cells infected with other SV40 strains. (
iv
) T, t, and 62-kilodalton polypeptides were readily labeled with [
35
S]methionine during a 10-min pulse; in a subsequent chase, the
35
S-labeled 94-kilodalton T antigen was apparently converted to 89- and 84-kilodalton polypeptides but not to either the 62-kilodalton polypeptide species or t antigen. (
v
) Partial peptide maps suggest that the 62-kilodalton polypeptide and T antigen are closely related. (
vi
) In addition to the above described 62-kilodalton polypeptide, a 54-kilodalton polypeptide was also detected. However, the 54-kilodalton species appears to be of cellular origin because it was immunoprecipitated with both normal and anti-T antiserum from uninfected and lytically infected cells and from virally transformed cells.
Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H.
Authors: Itoh T , Tomizawa J
Published: May 2006

A plasmid that consists of an 812-base-pair segment containing the replication origin of plasmid ColE1 and of a 1240-base-pair segment containing a beta-lactamase gene has been constructed. The plasmid DNA has three principal sites where transcription is initiated in vitro. One is located in the ColE1 segment 555 nucleotides upstream from the origin. Most transcription from this site extends past the origin; some of the transcripts form hybrids spontaneously with the template at their 3' portions. Cleavage of these transcripts by RNase H generates 3' termini at the origin region. When DNA polymerase I is included in the reaction along with RNA polymerase and RNase H, dAMP or dCMP is added directly onto the cleaved RNA molecules, most of which retain the intact 5' terminus. The addition of a deoxyribonucleotide to the cleaved RNA can be regarded as the first step of ColE1 DNA synthesis. Once it has served as a primer, the RNA is eliminated from the product by RNase H.
Density-dependent stimulation and inhibition of cell growth by agents that disrupt microtubules
Authors: McClain Donald A. , Edelman Gerald M.
Published: May 2006

We have previously reported that agents that disrupt microtubules, such as colchicine, inhibit the growth stimulation of lymphocytes and arrested fibroblasts; other workers have recently reported enhanced stimulation of fibroblasts in the presence of the same drugs. In the present studies, we resolve this conflict by demonstrating that the stimulatory and inhibitory effects of microtubule disruption depend upon the density and the cell type of the treated cultures. Our analysis included an examination of three variables: (
i
) cell density (sparse or confluent), (
ii
) cell type (resting fibroblasts from mouse or chicken embryos or from the permanent 3T3 mouse fibroblast line), and (
iii
) treatment with colchicine and related drugs in the presence or absence of various mitogens such as serum, insulin, and epidermal growth factor. We found that colchicine augmented mitogenesis in confluent cultures of all cell types. Colchicine by itself appeared to be mitogenic only for confluent chicken embryo fibroblasts. In sparse cultures with minimal cell-cell contacts, however, there were differences between embryonic cells and the 3T3 cell line. In confirmation of our previous reports, disruption of microtubules by colchicine inhibited the mitogenic stimulation of sparse cultures of embryonic chicken or mouse fibroblasts. In contrast, fibroblasts of the permanent 3T3 line in sparse cultures were stimulated by some mitogens despite the presence of colchicine. The augmentative effects of colchicine on the stimulation of confluent cultures were synergistic with the mitogens, and colchicine allowed response to otherwise submitogenic doses of growth factors. Kinetic studies indicated that the stimulatory and inhibitory effects of colchicine are separable and that both can operate simultaneously. The experiments suggest that the regulation of growth by nutrient deprivation and the regulation by density dependence proceed at least in part by different mechanisms. All of the results suggest that microtubular integrity can be associated with the expression of either negative or positive controls on cell growth, depending upon the confluence or lineage of the cells in culture.
Inheritance of acquired immunological tolerance to foreign histocompatibility antigens in mice.
Authors: Gorczynski R M , Steele E J
Published: May 2006

CBA mice were rendered tolerant of major histocompatibility antigens of A/J mice by neonatal injection of 100 x 10(6) lymphoid cells of (CBA X A/J)F1 followed by repeated injections of F1 cells at 2-week intervals throughout the study. When adult (8 weeks old), 10 tolerant or normal CBA males were mated to normal CBA females. Spleen cells of the progeny were tested for their ability to mount a cytotoxic T lymphocyte response in vitro against A/J antigens or against C57BL/6J and B10.A (2R) antigens in a cell-mediated lympholysis (CML) assay. A significant proportion (50-60%) of first-generation offspring of tolerant fathers failed to produce detectable anti-A/J cytotoxic responses but responded in the normal range to stimulation by C57BL/6J or B10.A (2R). Second-generation offspring derived from mating animals born of tolerant male parents--either brother x sister matings (incross or matings to normal CBA mice (outcross)--also showed a high proportion (20-40%) with diminished anti-A/J CML responses when similarly tested in vitro. Thus, a specific acquired somatic characteristic in the immune system (tolerance to major histocompatibility antigens) induced in male mice shows significant transmission to first- and second-generation offspring.
Isolation of variants of mouse myeloma X63 that express changed immunoglobulin class.
Authors: Radbruch A , Liesegang B , Rajewsky K
Published: May 2006

We have used fluorescence-activated cell sorting with class-specific antisera to isolate spontaneous variants in the expression of immunoglobulin heavy chain class from the mouse myeloma cell line X63 (IgGI, kappa). In the wild-type cell population, only one type of variants was found, namely, cells expressing IgG2b. From an IgG2b variant clone we isolated secondary variants that had either reverted to IgGI expression or expressed IgG2a or IgG2a and IgG2b concomitantly. The variant heavy chains are of normal size. The variant immunoglobulins were characterized serologically, and all of them still expressed the wild-type idiotype. Wild-type and variant cell populations were screened for heavy chain class-switch variants by fluorescence microscopy. A variety of switch variants was found in addition to the ones isolated by cell sorting, and a clear pattern of class switching (gamma 1 leads to gamma 2b leads to gamma 2a leads to alpha) with frequent reversion emerges from this analysis. Cells expressing new heavy chain classes occurred at frequencies of about 10(-7)-10(-6)/cell per generation, whereas revertants were as frequent as 10(-6)-10(-5)/cell per generation.
Heterogeneity of human high density lipoprotein: Presence of lipoproteins with and without apoE and their roles as substrates for lecithin:cholesterol acyltransferase reaction
Authors: Marcel Yves L. , Vezina Camilla , Emond Diane , Suzue Ginzaburo
Published: May 2006

By affinity chromatography on heparin-Sepharose, two classes of lipoproteins were separated from high density lipoproteins (HDL) isolated from patients with primary or secondary lecithin:cholesterol acyltransferase (LCATase; EC 2.3.1.43) deficiency and from normal subjects. The unretained fraction, HDL
A
, was characterized by having apoA-I as a major apoprotein; it also contained apoA-II, -C-II, and -C-III but it contained only traces of immunodetectable apoE and no apoB. The retained fraction, HDL
E
, had apoE as the major apoprotein; it also contained apoA-I, -A-II, -B, -C-II, and -C-III. The relative concentration of apoA-I increased with increasing density in the HDL
E
subclass. Compared to HDL
A
, HDL
E
had a significantly higher cholesterol content and a lower protein concentration. HDL
E
was mainly (90%) contained within the HDL
2
subfraction. Contamination of HDL
E
by low density lipoproteins (LDL) or Lp(a) was minimal on the basis of pre-β-electrophoretic mobility and absence of albumin, respectively. Contamination by LDL or Lp(a) could be resolved in part by application of HDL
E
to concanavalin A-Sepharose or to heparin-Sepharose with a shallow gradient. When evaluated as substrates for a highly purified LCATase preparation, the initial reaction rates and V
max
obtained with HDL
A
were always higher than those obtained with HDL
E
in any given plasma. However, both HDL subclasses from LCATase-deficient subjects were better substrates than the corresponding HDL subclasses from normal plasma. Also, both HDL
3A
and HDL
3E
isolated from normal HDL
3
were better substrates than the corresponding subclasses isolated from normal HDL
2
. The recognition of this compositional and functional heterogeneity within HDL will allow a better understanding of the metabolism of this lipoprotein class.
Cluster formation of opiate (enkephalin) receptors in neuroblastoma cells: differences between agonists and antagonists and possible relationships to biological functions.
Authors: Hazum E , Chang K J , Cuatrecasas P
Published: May 2006

Neuroblastoma cells were used to study the surface distribution and organization of opiate (enkephalin) receptors and the possible relevance of changes in these variables to biological functions. Opiate receptors readily form clusters that are visible by image-intensifier fluorescent microscopy and are localized on both the cell body and processes. These clusters do not become internalized even during prolonged incubation periods. The receptors appear to pre-exist largely in a diffuse state, with only a very small number pre-existing as clusters. The clusters are induced within 1 hr and they are stable for prolonged (7-9 hr) periods, even after removal of the receptor-bound ligand. Agonists and antagonists are both equally capable of inducing receptor clustering. However, the clusters induced by agonists are different from those induced by antagonists; the former can be dispersed by treatment with dithiothreitol. This dispersion requires removal of the receptor-bound agonist, indicating that the hormone protects or stabilizes disulfide bonds which are critical for maintenance of the clustered state. Pretreatment of cells with sulfhydryl-blocking reagents (iodoacetate, iodoacetamide, and N-ethylmaleimide) prevents cluster formation but does not alter the ability of agonists to inhibit adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity. Neither the number nor the affinity of binding sites is altered by pretreatment with opiates. These studies suggest that at least the acute, immediate biological effects of opiates and enkephalins occur prior to and are independent of the formation of gross receptor clusters. The possible relationship of cluster formation to the actions of opiates remains to be determined.