bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide sequence of the BK virus DNA segment encoding small t antigen.
Authors: Dhar R , Seif I , Khoury G
Published: May 2006

The nucleotide sequence from 0.64 to 0.53 map units in the BK virus genome coding for the small t protein has been determined. There is only one open reading frame that can code for a polypeptide of 172 amino acids, the putative small t protein. Beyond this segment, multiple termination codons are present in all three reading frames. There is considerable nucleotide and amino acid sequence homology between this region of BK virus and the analogous region of simian virus 40, especially in the proximal portion from 0.64 to 0.60 map units which is most likely common to the small t and large T BK virus proteins. A comparison of the conserved sequences within the early papovavirus genes both confirms the evolutionary relationship between these viruses and suggests the amino acid composition of the regions required for T antigen functions.
Mechanism of interferon action: Phosphorylation of protein synthesis initiation factor eIF-2 in interferon-treated human cells by a ribosome-associated kinase processing site specificity similar to hemin-regulated rabbit reticulocyte kinase
Authors: Samuel Charles E.
Published: May 2006

The phosphorylation of purified protein synthesis factors catalyzed by protein kinase preparations isolated from interferon-treated human amnion cells was examined. Ribosomal salt-wash fractions prepared from interferon-treated human cells contained a protein kinase that catalyzed the [γ-
32
P]ATP-mediated phosphorylation of the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2α); this kinase activity was significantly enhanced in interferon-treated as compared to untreated cells. The tryptic [
32
P]phosphopeptide pattern obtained for eIF-2α phosphorylated by the interferon-mediated human kinase was indistinguishable from the pattern obtained for eIF-2α phosphorylated by the hemin-regulated rabbit reticulocyte kinase when analyzed by thin-layer chromatography with three different solvent systems and by high-voltage electrophoresis.
O
-[
32
P]Phosphoserine was liberated by partial acid hydrolysis from eIF-2α phosphorylated by either the human or the rabbit kinase. In addition to the phosphorylation of eIF-2α, interferon treatment of human cells enhanced the phosphorylation of two additional ribosome-associated proteins designated P
1
and P
f
. The major phosphoester linkage observed for the human, as well as murine, phosphoprotein P
1
was
O
-phosphoserine. The interferon-mediated phosphorylation of both eIF-2α and protein P
1
was dependent upon the presence of RNA with double-stranded character; P
f
phosphorylation was not affected by double-stranded RNA. These results suggest that the interferon-mediated ribosome-associated human protein kinase catalyzes the phosphorylation of eIF-2α in a site-specific manner that is apparently identical with the reaction catalyzed by the hemin-regulated rabbit reticulocyte kinase; hence, the phosphorylation of eIF-2 may play a role in regulating the initiation of translation in interferon-treated cells.
Enzymatic activities associated with a purified simian virus 40 T antigen-related protein
Authors: Tjian R. , Robbins A.
Published: May 2006

A protein antigenically related to the simian virus (SV 40)
A
gene product has been purified to near homogeneity from cells infected with the adenovirus-SV 40 hybrid virus Ad2
+
D2 and shown to contain ATPase (ATP phosphohydrolase, EC 3.6.1.3) and protein kinase (ATP:phosphotransferase, EC 2.7.1.37) activity. Both enzymatic activities copurify with the protein through six stages including one gel filtration column, two ion exchange columns, and a heparin affinity column. Analogous fractions from extracts of cells uninfected or infected with adenovirus 2 alone do not contain these enzymatic activities. The D2 hybrid protein resolves into two forms (I and II) during ion exchange chromatography. Form I, the major species (85%) of the D2 hybrid protein, elutes from DEAE-Sephadex in 0.37 M NaCl and is able to catalyze the hydrolysis of ATP to ADP + P
i
at a rate of 3 μmol/hr per mg. The remaining 10-15% of the D2 hybrid protein consists of form II which elutes from DEAE-Sephadex in 0.29 M NaCl and is able to hydrolyze ATP as well as to incorporate phosphorus from ATP into either the D2 hybrid protein itself or other protein acceptors such as phosvitin. Although both forms are able to bind DNA, the ATPase activity of form I cosediments with SV 40 DNA more efficiently than does the protein kinase activity of form II during glycerol gradient centrifugation. The ATPase activity of form I is efficiently inhibited by addition of anti-T gamma globulin to the reaction mixture whereas control gamma globulin has no effect. Similarly, the phosphorylation of the D2 hybrid protein by form II is inhibited by anti-T gamma globulin. By contrast, phosphorylation of phosvitin is specifically inhibited by antibody only when the immune complex is removed from the reaction mixture. Thus, it appears likely that one and possibly two enzymatic activities are carried out by the D2 hybrid protein. These findings are discussed in terms of mechanisms of SV 40 DNA replication and virally induced transformation.
Effect of benzo[a]pyrene-diolepoxide on infectivity and in vitro translation of phage MS2 RNA.
Authors: Sagher D , Harvey R G , Hsu W T , Weiss S B
Published: May 2006

Previous studies have shown that alkylation of MS2 RNA by certain derivatives of polycyclic aromatic hydrocarbons renders it noninfectious. Since phage RNA serves as a template for translation and transcription, either of these RNA-directed processes, or both, could be responsible in vivo for the inhibition of phage replication by metabolically activated hydrocarbons. The present study correlates the degree of inhibition of MS2 RNA infectivity, at various levels of alkylation by (+/-)-trans, 7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzol[a]pyrene, with the translation efficiency in vitro of the same alkylated RNA for the synthesis of viral synthetase and of maturation and coat proteins. The results indicate that dihydroxyepoxy-tetrahydrobenzo[a]pyrene modification of MS2 RNA impairs its template capacity for the synthesis of phage-specific proteins; this inhibition is insufficient, however, to account for the loss of RNA infectivity at lower molar ratios of alkylation. For the three viral proteins synthesized in vitro, the translation of RNA synthetase is much more sensitive to MS2 RNA modification than either coat or maturation protein synthesis. Our results also indicate that the loss of viral RNA infectivity follows a single-hit inactivation mechanism, whereas several alkylation events in the viral RNA synthetase cistron may be necessary to block translation of this gene product.
Initial site of insulin cleavage by insulin protease.
Authors: Duckworth W C , Stentz F B , Heinemann M , Kitabchi A E
Published: May 2006

Exposure of insulin to insulin protease (insulinase, EC 3.4.22.11), a degradative enzyme with considerable specificity toward insulin, results in alterations in the properties of the insulin molecule. Limited degradation by the enzyme results in a decrease in the ability of insulin to bind to membrane receptors with less change in the immunoprecipitability or trichloracetic acid precipitability of the hormone. Limited degradation by insulin protease also alters insulin so that the molecule becomes susceptible to attack by nonspecific endopeptidases which have no effect on unaltered insulin. These data demonstrate the production of an intermediate in the proteolytic degradation of insulin. By labeling with [14C]dansyl chloride, an insulin intermediate with three amino-terminal residues, glycine, phenylalanine, and leucine, was identified. Analysis of this intermediate demonstrated that it was composed of an intact A chain and a B chain cleaved between residues B16 and B17, with the three peptide chains held together by disulfide bonds. Based on these findings, we hypothesize that a stepwise degradation of insulin occurs in vivo and that an early step in the process is the cleavage between B16 and B17 that renders the molecule sucseptible to further degradation by nonspecific proteases.
Localized deposition of chitin on the yeast cell surface in response to mating pheromone
Authors: Schekman Randy , Brawley Vicki
Published: May 2006

Treatment of
a
mating-type
Saccharomyces cerevisiae
cells with the pheromone α-factor (secreted by α mating-type cells) induces the synthesis of chitin. Small daughter cells, which start with no detectable chitin, make 3 times more chitin when grown in the presence of α-factor than do untreated exponentially growing cells. Budding cells accumulate chitin in the nascent division septum [Cabib, E. & Bowers, B. (1975)
J. Bacteriol
. 124, 1586), as detected by staining with the fluorescent dye primulin. In the absence of a division septum, α-factor-treated cells accumulate chitin in the area of pheromone-stimulated growth. Enzymatic lysis of budding and pheromone-treated cells allows the separation of membrane-bound chitin synthase (UDP-2-acetamido-2-deoxy-D-glucose: chitin 4-β-acetamidodeoxyglucosyltransferase, EC 2.4.1.16) activity from a dense particulate fraction containing chitin. Chitin synthase activity is associated with both the plasma membrane and small intracellular particles. During pheromone treatment, the levels of chitin synthase in the plasma membrane and in intracellular particle fractions increase 11- and 4-fold, respectively. Although chitin synthase is made as zymogen that requires proteolytic activation, the plasma membrane of pheromone-treated cells shows a significant fraction of preactivated enzyme; intracellular membrane-bound synthase is found exclusively in the zymogen form.
Altered pattern of growth and differentiation in human keratinocytes infected by simian virus 40.
Authors: Steinberg M L , Defendi V
Published: May 2006

Human epidermal keratinocytes were infected by simian virus 40 in vitro. The structure of the developing keratinocyte colony reflects the spatial separation of cell division and keratinization in intact skin; thymidine-incorporating cells were primarily localized at the colony periphery whereas nondividing, histologically differentiated cells accumulated in the interior. Viral infection produced a dramatic increase in the size of the proliferative population as, simultaneously, differentiation was reduced in the colony interior. These changes were manifest when simian virus 40 T-antigen synthesis was detectable in only a small percentage of the cells; differentiation became increasingly density dependent as the percentage of T-antigen-positive cells rose over serial passage. The disruption of the normal pattern of growth/differentiation localization coincided with a loss of dependence on serum for growth, but preceded the appearance of other virus-induced properties associated with transformation; i.e., the ability to form colonies in soft agar and independence of growth from fibroblasts.
Electrical currents through full-grown and maturing Xenopus oocytes.
Authors: Robinson K R
Published: May 2006

An extracellular vibrating electrode was used to map the current pattern around Xenopus laevis oocytes. Current was found to enter the animal hemisphere and leave the vegetal hemisphere; in fully grown oocytes from which the follicle cells had been removed, the maximal current density was about 1 microamperemeter/cm2. This current decreased to nearly zero in response to progesterone and several other maturation-producing agents. In the case of progesterone, the decline began within a few minutes of the addition of the hormone and proceeded with a half-time of about 20 min. An analysis of the effects on the current of the removal or addition of various ions and drugs led to the inference that the major current-carrying ion was chloride and that the chloride permeability was controlled by calcium.
Changes in quaternary structure of IgG upon reduction of the interheavy-chain disulfide bond.
Authors: Seegan G W , Smith C A , Schumaker V N
Published: May 2006

Reduction of the single disulfide bond between heavy chains in the hinge region of rabbit IgG antibody causes destabilization of the CH2 region of the molecule. Our studies indicate that reduced antibody molecules undergo a large change in quaternary structure in the CH2 region upon aggregation with a small bivalent hapten. The conformational change was observed both in hydrodynamic studies and by electron microscopy. The sizes of native and reduced antibody complexes were measured from electron micrographs. These measurements show that reduction of the hinge disulfide allows the CH2 domains of the antibody to separate under the strain induced by complex formation. The Fab arms, which are clearly seen in the electron micrographs of the native complexes, are extended by a portion of the Fc region to effectively become Facb arms in the reduced complexes. The length of the arms is effectively increased by 23 A. This results in a massive alteration in the quaternary structure of the CH2 region of the molecule, and this may be the basis of many of the effects of mild reduction on the various effector functions of the antibody molecule. These findings also support the open structure of the CH2 region proposed on the basis of crystallographic analyses, and they demonstrate how the interheavy-chain hinge disulfide restricts segmental flexibility in the Fc fragment of the IgG molecule.
Triene prostaglandins: Prostacyclin and thromboxane biosynthesis and unique biological properties
Authors: Needleman Philip , Raz Amiram , Minkes Mark S. , Ferrendelli James A. , Sprecher Howard
Published: May 2006

Platelets enzymatically convert prostaglandin H
3
(PGH
3
) into thromboxane A
3
. Both PGH
2
and thromboxane A
2
aggregate human platelet-rich plasma. In contrast, PGH
3
and thromboxane A
3
do not. PGH
3
and thromboxane A
3
increase platelet cyclic AMP in platelet-rich plasma and thereby: (
i
) inhibit aggregation by other agonists, (
ii
) block the ADP-induced release reaction, and (
iii
) suppress platelet phospholipase-A
2
activity or events leading to its activation. PGI
3
(Δ
17
-prostacyclin; synthesized from PGH
3
by blood vessel enzyme) and PGI
2
(prostacyclin) exert similar effects. Both compounds are potent coronary relaxants that also inhibit aggregation in human platelet-rich plasma and increase platelet adenylate cyclase activity. Radioactive eicosapentaenoate and arachidonate are readily and comparably acylated into platelet phospholipids. In addition, stimulation of prelabeled platelets with thrombin releases comparable amounts of eicosapentaenoate and arachidonate, respectively. Although eicosapentaenoic acid is a relatively poor substrate for platelet cyclooxygenase, it appears to have a high binding affinity and thereby inhibits arachidonic acid conversion by platelet cyclooxygenase and lipoxygenase. It is therefore possible that the triene prostaglandins are potential antithrombotic agents because their precursor fatty acids, as well as their transformation products, PGH
3
, thromboxane A
3
, and PGI
3
, are capable of interfering with aggregation of platelets in platelet-rich plasma.
Localization of the decoding region on the 30S Escherichia coli ribosomal subunit by affinity immunoelectron microscopy.
Authors: Keren-Zur M , Boublik M , Ofengand J
Published: May 2006

The decoding region of the Escherichia coli ribosome has been localized by affinity immunoelectron microscopy. Valyl-tRNA1Val, derivatized at the alpha-amino end with the dinitrophenyl group, was bound to the ribosomal P site of 70S ribosomes and crosslinked specifically to 16S RNA by 310- to 325-nm irradiation. Previous work has shown that crosslink occurs between the 5' anticodon base of the tRNA and a pyrimidine in the 3' one-third of the 16S RNA. By reaction of the dinitrophenyl group with antibody, dimers of the 30S tRNA covalent complexes were prepared containing one covalently attached tRNA per 30S subunit. Electron microscopic visualization of the antibody attached to the dinitrophenyl group located the position of the 3' end of the tRNA. Several sites, with a strong preference for the large protrusion or cleft region in the upper one-third of the subunit, were found. The multiplicity of sites is likely due to the freedom of orientation of the 3' end of the tRNA which is approximately 80 A from the site of crosslinking. By extrapolating this distance from each of the antigenic sites, we concluded that the anticodon of tRNA when in the P site is probably located in the cleft region of the 30S subunit.
Enzymatic insertion of purine bases into depurinated DNA in vitro.
Authors: Livneh Z , Elad D , Sperling J
Published: May 2006

An enzymatic activity that inserts purines into depurinated DNA was found in a soluble enzyme extract of Escherichia coli. This activity brings about the insertion of adenine and guanine into the appropriate apurinic sites in double-stranded DNA by using the corresponding deoxyribonucleoside triphosphates as the purine donors. Magnesium ions are required for this activity, it is inhibited by caffeine, and it does not act on depurinated single-stranded DNA. The insertion activity described here may represent a step in a repair mechanism, "base-insertion repair," whereby apurinic sites (which may occur in double-stranded DNA either due to the removal of damaged purines with specific glycosylases or by spontaneous depurination) are directly filled with the correct missing purine base.
Escherichia coli adenylate cyclase complex: regulation by the proton electrochemical gradient.
Authors: Peterkofsky A , Gazdar C
Published: May 2006

Sugars such as glucose are transported into Escherichia coli by a coupled phosphorylation mechanism (the phosphoenolpyruvate:sugar phosphotransferase system, PTS). Transport of sugars through the PTS results in inhibition of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity by a mechanism involving a change in the state of phosphorylation of PTS proteins. Other sugars (e.g., lactose) are transported without modification by a mechanism involving proton cotransport, which requires a proton motive force across the cell membrane. We show here that uptake of sugars through the lactose transport system results in inhibition of adenylate cyclase activity if the proton symport mechanism is also active. The protonophore carbonyl cyanide m-chlorophenylhydrazone also inhibits adenylate cyclase activity. These data suggest that the steady-state electrochemical proton gradient regulates the activity of adenylate cyclase. We propose that sugar-dependent inhibition of adenylate cyclase activity may occur by either of two mechanisms. Sugars transported by the PTS inhibited adenylate cyclase activity by dephosphorylation of a regulatory protein, while sugars transported by the proton motive force system inhibit adenylate cyclase activity as a result of collapse of the proton electrochemical gradient.
Rapid helix--coil transitions in the S-2 region of myosin.
Authors: Tsong T Y , Karr T , Harrington W F
Published: May 2006

Temperature-jump studies on the long S-2 fragment (100,000 daltons) isolated from myosin show that this structure can undergo alpha-helix--random coil transitions in a time range approximating the cycle time of a crossbridge. Two relaxation times are observed after temperature jumps of 5 degrees C over the range 35--55 degrees C, one in the submillisecond (tau f) and the other in the millisecond (tau s) time ranges. Both processes exhibit maxima near the midpoint of the helix--coil transition (tm = 45 +/- 2 degrees C) as determined by optical rotation melt experiments. Similar results were observed for the low temperature transition (tm = 45 degrees C) of the myosin rod. Viscosity studies reveal that the S-2 particles has significant flexibility at physiological temperature. Results are considered in terms of the Huxley--Simmons and helix--coil transition models for force generation in muscle.
3'-Terminal nucleotide sequence of alfalfa mosaic virus RNA 4.
Authors: Koper-Zwarthoff E C , Bol J F
Published: May 2006

The sequence of the 3'-terminal 91 nucleotides of alfalfa mosaic virus RNA 4, the messenger for the viral coat protein, has been elucidated. A fragment containing the 3' terminus of the RNA was obtained by mild digestion with RNase T1. The primary structure of the fragment was deduced by labeling it in vitro at its 5' terminus and application of RNA sequencing techniques. The sequence is completely extracistronic and is believed to contain the binding sites for the viral coat protein and replicase.
Preparation of a disulfide-linked dimer of human placental lactogen fragment 1--134 with immunologic and biologic activity.
Authors: Russell J , Sherwood L M , Kowalski K , Schneider A B
Published: May 2006

In order to determine the chemical features of human placental lactogen (hPL) necessary for its biologic activity we prepared the following fragments from the plasmin-cleaved hormone: reduced and alkylated 1--134, reduced and alkylated 141--191, and a 1--134 dimer joined through the single cysteinyl residue at position 53. In a radioimmunoassay using antibodies against native hPL, the two reduced and alkylated fragments produced nonparallel displacement and had less than 1% of the activity of hPL. The ability of reduced and alkylated 1--134 to bind to mammary gland receptors was less than 5% of that of hPL; reduced and alkylated 141--191 showed no detectable activity in the same assay. The 1--134 dimer, in contrast, had 20% of the immunologic activity and 30% of the ability to bind to lactogenic receptors relative to the native hormone. In an in vitro bioassay the lactogenic activity of the 1--134 dimer was equivalent to that of the native hormone. The circular dichroic spectra of hPL, reduced and alkylated 1--134, and 1--134 dimer indicated that the dimer had regained much of the helical content of the native hormone. Antibodies produced to reduced and alkylated 1--134 did not significantly crossreact with either native hPL or 1--134 dimer. From these data we conclude that the information for the lactogenic activity of hPL is contained in the first 134 amino acid residues and that the proper conformation is necessary for its biologic expression.
Simultaneous localization of type II collagen and core protein of chondroitin sulfate proteoglycan in individual chondrocytes.
Authors: Vertel B M , Dorfman A
Published: May 2006

In order to investigate the coordinated synthesis of matrix components by individual chondrocytes, specific antibodies to type I collagen, type II collagen, and chondroitin sulfate proteoglycan core protein were used in simultaneous double immunofluorescence reactions. Extensive accumulation of core protein surrounding chondrocytes and the intracellular accumulation of type II collagen were observed. Extracellular core protein immunofluorescence obscured the intracellular reaction product, but the extracellular immunoreactive material could be removed by digestion with purified testicular hyaluronidase prior to fixation. Subsequent to digestion, core protein and type II collagen were observed in the same chondrocytes within discrete, sometimes identical, cytoplasmic regions, thus demonstrating the simultaneous localization of these two products characteristic of differentiating cartilage.
Induction of terminal differentiation in human promyelocytic leukemia cells by tumor-promoting agents.
Authors: Huberman E , Callaham M F
Published: May 2006

Human promyelocytic leukemia cells (HL-60) were induced to differentiate into mature cells by the tumor-promoting agent phorbol-12-myristate-13-acetate and other related phorbol diesters. Differentiation was determined by an increase in the percent of myelocytes, metamyelocytes, and other mature myeloid cells as well as by an increase in the percent of phagocytizing cells. Induction of differentiation could be determined after 2 days of treatment with phorbol-12-myristate-13-acetate at a dose as low as 6 X 10(11) M. A correlation was found between reported tumor-promoting activity of a series of phorbol esters and their ability to induce myeloid differentiation and to inhibit cell growth. It is suggested that tumor-promoting agents like chemicals that induce terminal differentiation in these cells, at extremely low concentrations, may be used as a tool in the study of the control of cell growth, cell differentiation, and malignancy in human leukemic cells.
DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells.
Authors: Wigler M , Pellicer A , Silverstein S , Axel R , Urlaub G , Chasin L
Published: May 2006

In this report, we demonstrate the feasibility of transforming mouse cells deficient in adenine phosphoribosyltransferase (aprt; AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) to the aprt+ phenotype by means of DNA-mediated gene transfer. Transformation was effected by using unfractionated high molecular weight genomic DNA from Chinese hamster, human, and mouse cells and restriction endonuclease-digested DNA from rabbit liver. The transformation frequency observed was between 1 and 10 colonies per 10(6) cells per 20 microgram of donor DNA. Transformants displayed enzymatic activity that was donor derived as demonstrated by isoelectric focusing of cytoplasmic extracts. These transformants fall into two classes: those that are phenotypically stable when grown in the absence of selective pressure and those that are phenotypically unstable under the same conditions.
Biosynthesis
in vitro
of immunoreactive 31,000-dalton corticotropin/β-endorphin-like material by bovine hypothalamus
Authors: Liotta Anthony S. , Gildersleeve David , Brownstein Michael J. , Krieger Dorothy T.
Published: May 2006

Enzymatically dispersed bovine hypothalamic or cortical tissue was maintained in culture in the presence of
3
H-labeled amino acids. After such incubation, extracts of cells and of media contained
3
H-labeled products that were specifically bound by immobilized affinity-purified antisera to corticotropin (ACTH) and β-endorphin. The majority of these products eluted in the void volume (
V
0
) upon Sephadex G-50 gel filtration; minor
3
H-labeled products eluted in the regions of the ovine β-lipotropin marker and in fractions having apparent molecular weights of approximately 12,000 and 3800. Sequential use of these immobilized antisera revealed that most of the
V
0
material contained both ACTH and β-endorphin antigenic determinants within the same molecule(s), whereas retarded material contained only one of the determinants. When this
V
0
material was rerun on a Sephadex G-75 column, it coeluted with the 31-kilodalton precursor of both ACTH and β-endorphin obtained from a bovine anterior pituitary extract. Thus, the high molecular weight immunoreactive ACTH/β-endorphin-like
3
H-labeled product(s) derived from the hypothalamic culture is similar to the pituitary-derived precursor in containing the dual antigenic determinants and in its gel filtration characteristics. In contrast, the cortex-derived cell preparation was devoid of
3
H-labeled products specifically reactive with the antisera employed.
A peptide as a possible transmitter in sympathetic ganglia of the frog.
Authors: Jan Y N , Jan L Y , Kuffler S W
Published: May 2006

A search was made in sympathetic ganglia of the bullfrog for a noncholinergic, nonaminergic transmitter that is released by a distinct group of preganglionic axons. These initiate a late slow excitatory postsynaptic potential which lasts for many minutes. The most promising candidate for the role of transmitter is a peptide that resembles luteinizing hormone-releasing hormone (LHRH; luliberin). The reasons are: (i) LHRH (1 muM) and some of its analogs cause a slow depolarization of ganglion cells. (ii) Radioimmunoassays established that 100-800 pg of a LHRH-like substance is contained in the lumbar chain of sympathetic ganglia. (iii) The LHRH-like material is specifically distributed in those spinal nerves that contain axons that initiate the slow noncholinergic synaptic responses. (iv) Five days after ipsilateral preganglionic axons are cut, 95% of the LHRH-like substance disappears from ganglia, while the LHRH immunoreactivity triples in the spinal nerves proximal to the cut region. (v) About 0.6% of the LHRH-like material within ganglia can be collected from the perfusate after 30 min of incubation in isotonic KC1; this release is Ca dependent. (vi) The candidate for transmitter has several chemical characteristics of a peptide and has a Mr near 1000.
Regulation of protein synthesis in reticulocyte lysates: immune serum inhibits heme-regulated protein kinase activity and differentiates heme-regulated protein kinase from double-stranded RNA-induced protein kinase.
Authors: Petryshyn R , Trachsel H , London I M
Published: May 2006

A specific immune serum to the heme-regulated inhibitor (HRI) has been prepared by immunizing chickens with highly purified reversible HRI prepared from rabbit reticulocyte lysates. Studies with this immune serum demonstrate that the behavior of purified reversible HRI is similar to that of the inhibitor activated in rabbit reticulocyte lysates: the immune serum (i) inhibits the phosphorylation of the small subunit (38,000 daltons) of the eukaryotic initiation factor eIF-2 by both crude and purified inhibitor preparations; (ii) prevents the concomitant inhibition of protein synthesis by both crude and purified inhibitor preparations; and (iii) prevents the autophosphorylation of the 95,000-dalton polypeptide in purified and crude HRI preparations. The protein kinase and inhibitory activities of crude and partially purified preparations of the double-stranded RNA-induced inhibitor of protein synthesis are not affected by the immune serum prepared to reversible HRI. These results indicate that the inhibitor induced by double-stranded RNA is antigenically distinct from the reversible HRI.
Regulation of DNA replication in S phase nuclei by ATP and ADP pools
Authors: Rapaport Eliezer , Garcia-Blanco Mariano A. , Zamecnik Paul C.
Published: May 2006

Synchronized 3T6 (mouse fibroblast) ghost monolayers (isolated nuclei) were utilized to study the effects of ATP and ADP levels on DNA replication
in vitro
. A system yielding discontinuous semiconservative DNA replication (without any detectable repair) in synchronized S phase nuclei has been developed. Lack of initiation of new sites has been observed in isolated S phase 3T6 nuclei without the presence of cytoplasmic material; DNA synthesis is comprised only of elongation at sites where initiation had previously taken place. DNA synthesis in S phase nuclei proceeded optimally at an ATP concentration of 4-5 mM. High ATP levels as well as high ATP/ADP ratios (produced by an ATP-regenerating system at a variety of ATP concentrations) yielded marked inhibition of [
3
H]dTTP incorporation. The cellular and nuclear pools of ATP and ADP in intact synchronized 3T6 cells were accurately determined by high-pressure liquid chromatography. A good correlation with the studies on isolated nuclei has been observed. Whereas total cellular ATP pools increase during the progression of 3T6 cells from G
1
to S phase of the cell cycle, nuclear ATP pools do not increase, and the nuclear ATP/ADP ratios decrease once the cells enter the S phase of their cycle. These experiments suggest that nuclear ATP pools and ATP/ADP ratios act as S phase controls, regulating DNA elongation at sites where its synthesis has previously been initiated by cytoplasmic factors.
Further studies on the mode of action of the heme-controlled translational inhibitor.
Authors: de Haro C , Ochoa S
Published: May 2006

We have isolated [de Haro, C. & Ochoa, S. (1978) Proc. Natl. Acad. Sci. USA 75, 2713-2716] a protein factor (eIF-2 stimulating protein, ESP) that is essential for formation of ternary and 40S initiation complexes by the eukaryotic polypeptide chain initiation factor 2 (eIF-2) at the low concentrations of eIF-2 present in reticulocyte lysates. The fact that stimulation of complex formation by ESP is virtually abolished when the small (38,000 daltons) subunit of eIF-2 is phosphorylated by ATP in the presence of eIF-2 kinase (heme-controlled inhibitor, HCI) is consistent with the notion that HCI inhibits translation in lysates by blocking the interaction of eIF-2 with ESP. Our present work, with highly purified eIF-2 and ESP, has additionally established that, unlike phosphorylation of the small subunit, phosphorylation of the middle (52,000 daltons) subunit of eIF-2, which does not lead to translational inhibition in lysates, does not affect eIF-2-ESP interaction. This provides further support for our model of translational inhibition by HCI.
Stimulation of Ca
2+
-dependent neurotransmitter release and presynaptic nerve terminal protein phosphorylation by calmodulin and a calmodulin-like protein isolated from synaptic vesicles
Authors: Delorenzo Robert J. , Freedman Steven D. , Yohe Wendy B. , Maurer Steven C.
Published: May 2006

Synaptic vesicles have a Ca
2+
-dependent protein kinase system that may play a role in mediating Ca
2+
-stimulated neurotransmitter release and vesicle function. Calcium's ability to initiate norepinephrine release and protein phosphorylation in synaptic vesicle preparations was shown to be stimulated by the presence of an endogenous heat-stable vesicle protein fraction. The heat stability and characteristics of this endogenous vesicle fraction were similar to those of calmodulin (Ca
2+
-dependent regular protein) isolated from rat and bovine brain. Calmodulin, like endogenous heat-stable vesicle factor, restored calcium's ability to stimulate vesicle neurotransmitter release and protein kinase activity. Calmodulin-like vesicle protein and purified calmodulin were also equally effective in stimulating cyclic nucleotide-dependent phosphodiesterase, further indicating that these two proteins are functionally equivalent. Depolarization-dependent Ca
2+
uptake in intact synaptosomes simultaneously stimulated release of neurotransmitter and phosphorylation of particular synaptic vesicle proteins that were shown in the isolated vesicle preparation to be dependent on Ca
2+
and calmodulin. The results suggest that calcium's effects on neurotransmitter release and presynaptic nerve terminal protein phosphorylation may be mediated by endogenous calmodulin-like proteins.
Most myosin heavy chain mRNA in L6E9 rat myotubes has a short poly(A) tail.
Authors: Benoff S , Nadal-Ginard B
Published: May 2006

The mRNA for rat muscle myosin heavy chain (MHC) was isolated from L6E9 myotubes by two rounds of sucrose density gradient centrifugation followed by fractionation on an agarose/polyacrylamide gel. The purity of the mRNA isolated was determined by translation in vitro, peptide analysis of the in vitro product and comparison with authentic MHC, analysis of the kinetics of hybridization with cDNA prepared with this RNA, and titration analysis of total cytoplasmic RNA from muscle and nonmuscle sources. By using the MHC cDNA as probe of myogenic differentiation, it was observed that the level of cytoplasmic MHC mRNA increased approximately 200-fold as the dividing myoblast differentiated into the fused myotube. Titration analysis of RNAs fractionated by oligo(dT)-cellulose chromatography indicated that the majority of the increase occurred in that RNA population that failed to bind to an oligo(dT)-cellulose column.
Isolation of lysosomal α-mannosidase mutants of Chinese hamster ovary cells
Authors: Robbins April R.
Published: May 2006

A method is presented for detection of lysosomal hydrolase activity
in situ
in colonies of Chinese hamster ovary cells. This method was used to screen for mutants deficient in lysosomal α-mannosidase. Mutagenized cells were replicated onto filter papers [Esko, J. D. & Raetz, C. R. H. (1978)
Proc. Natl. Acad. Sci. USA
75, 1190-1193] and allowed to divide for 11-14 days; cells on the filter replicas were lysed, and the filters were incubated with 4-methylumbelliferyl-α-D-mannopyranoside at pH 4.4 and then photographed under UV light. Colonies for which the replicas exhibited little or no fluorescence were picked from the master plates and purified; mutants with decreased α-mannosidase activity were obtained at a frequency of 1 per 1500 mutagenized cells. Analysis of one of these mutants showed that lysosomal α-mannosidase activity was 18% of that from wild-type cells, whereas the activities of α-mannosidases not of lysosomal origin were similar in the wild type and mutant (these included both Golgi-associated and cytosolic enzymes as well as a novel acid α-mannosidase seen only in cells from confluent culture). The mutant contained normal levels of other lysosomal hydrolases. Both wild-type and mutant cells secreted α-mannosidase into the medium at levels proportional to those found inside the cells. Incubation of mutant cells with secretions from the wild type resulted in partial restoration of enzyme activity. Added enzyme was localized in the lysosomal fractions; uptake of added enzyme was inhibited by mannose 6-phosphate and fructose 1-phosphate, which are known to inhibit uptake of lysosomal enzymes into human diploid fibroblasts.
Thyrotropin is not a growth factor for human thyroid cells in culture
Authors: Westermark Bengt , Karlsson F. Anders , Wålinder Olov
Published: May 2006

Thyroid cells, obtained from both normal human tissue and benign nodular goiter, were cultured and maintained
in vitro
in 4-18 passages. Cultures with confluent cells accumulated cyclic AMP (10-150 times the basal amount) upon addition of bovine thyrotropin (100 milliunits/ml), indicating that the cells in culture maintained a thyrotropin-sensitive adenylate cyclase system. Addition of high doses of thyrotropin also induced a characteristic and reversible change in the morphology of the cells.

The effect of thyrotropin on cell growth was studied in short- and long-term experiments. Thyrotropin reduced [
3
H]thymidine incorporation in a dose-dependent fashion in all cultures of thyroid cells. The maximal inhibition over a 24-hr period was about 50%. The thyroid cells were notably sensitive, and the half-maximal effect occurred at about 100 milliunits of thyrotropin per ml. In contrast, the hormone had no effect on [
3
H]-thymidine incorporation into human glial cells. Low doses of thyrotropin also had no effect on human fibroblasts and, at high doses, a stimulation of [
3
H]thymidine incorporation was seen. Thyroid cell cultures grown in the presence of 10 milliunits of thyrotropin per ml for 7-14 days had a slower growth rate and 24-36% lower cell numbers at saturation density than control dishes, indicating that the hormone also had a long-term effect on cell proliferation. The data agree with
in vitro
studies by others of the effects of corticotropin and lutropin on target cells and suggest that
in vivo
the primary action of pituitary trophic hormones on endocrine tissues is not stimulation of growth.
S
-Adenosylhomocysteine toxicity in normal and adenosine kinase-deficient lymphoblasts of human origin
Authors: Kredich Nicholas M. , Hershfield Michael S.
Published: May 2006

The human lymphoblast line WI-L2 is subject to growth inhibition by a combination of the adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4.) inhibitor
erythro
-9-(2-hydroxy-3-nonyl)adenine (EHNA) and adenosine. Although adenosine-induced pyrimidine starvation appears to contribute to this effect, uridine only partially reverses adenosine toxicity in WI-L2 and not at all in strain 107, an adenosine kinase-(ATP:adenosine 5′-phosphotransferase, EC 2.7.1.20) deficient derivative of WI-L2. Treatment of both cell lines with EHNA and adenosine leads to striking elevations in intracellular
S
-adenosyl-L-homocysteine (AdoHcy), a potent inhibitor of
S
-adenosyl-L-methionine (AdoMet)-dependent methylation reactions. The methylation
in vivo
of both DNA and RNA is inhibited by concentrations of EHNA and adenosine that elevate intracellular AdoHcy. Addition of 100 μM L-homocysteine thiolactone to cells treated with EHNA and adenosine enhances adenosine toxicity and further elevates AdoHcy to levels approximately 60-fold higher than those obtained in the absence of this amino acid, presumably by combining with adenosine to form AdoHcy in a reaction catalyzed by
S
-adenosylhomocysteine hydrolase (EC 3.3.1.1). In the adenosine kinase-deficient strain 107, a combination of ADA inhibition and L-homocysteine thiolactone markedly increases intracellular AdoHcy and inhibits growth even in the absence of exogenous adenosine. These results demonstrate a form of toxicity from endogenously produced adenosine and support the view that AdoHcy, by inhibiting methylation, is a mediator of uridine-resistant adenosine toxicity in these human lymphoblast lines. Furthermore, they suggest that AdoHcy may play a role in the pathogenesis of the severe combined immunodeficiency disease found in most children with heritable ADA deficiency.
Specific binding of perhydrohistrionicotoxin to Torpedo acetylcholine receptor.
Authors: Elliott J , Dunn S M , Blanchard S G , Raftery M A
Published: May 2006

Torpedo californica postsynaptic membrane fragments were treated with base, which resulted in membranes that were depleted of many nonacetylcholine receptor polypeptides and contained acetylcholine receptor subunits of Mr 40,000, 50,000, 60,000, and 65,000 (Raftery, M.A., Vandlen, R.L., Reed, K.L. & Lee T. (1975) Cold Spring Harbor Symp. Quant. Biol. 40, 193-202). A 43,000-Mr polypeptide and some other components were quantitatively extracted. Base-treated membranes retained the capacity to bind [3H]perhydrohistrionicotoxin and the local anesthetics dibucaine and tetracaine. The regulation of this binding by carbamylcholine, as well as the kinetic mechanism of perhydrohistrionicotoxin binding, was unchanged. [3H]Perhydrohistrionicotoxin binding activity was largely reconstituted from 2% sodium cholate extracts of base-treated membranes. Therefore, the perhydrostrionicotoxin binding site appears to be located on one or more of the acetylcholine receptor polypeptides, and the reconstitution of that binding site from detergent extracts does not require the presence of a 43,000-Mr polypeptide.
Circular dichroism and magnetic circular dichroism of nitrogenase proteins
Authors: Stephens P. J. , McKenna C. E. , Smith B. E. , Nguyen H. T. , McKenna M-C. , Thomson A. J. , Devlin F. , Jones J. B.
Published: May 2006

Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of nitrogenase components (MoFe protein and Fe protein) from
Azotobacter vinelandii
(Av) and
Klebsiella pneumoniae
(Kp) have been obtained in the near infrared—visible—near ultraviolet spectral region. Previously, visible CD was reported to be absent or barely detectable in nitrogenase proteins; MCD spectra have not been reported. The chiroptical spectra can be measured in solution at room temperature, an advantage relative to spectroscopic methods requiring cryogenic sample temperatures. Absorption spectra were also obtained. The CD and MCD are markedly more structured, and thus interpretively more useful, than the corresponding absorption spectra. The dithionite-reduced MoFe proteins (Av1, Kp1) have nearly identical CD and MCD, demonstrating identical numbers and types of metal centers in similar protein environments. The CD and MCD cannot be explained solely in terms of contributions from known 4-Fe or 2-Fe clusters; the near-infrared MCD is inconsistent with the presence of known 4-Fe clusters. CD and MCD spectra of Lauth's violet-oxidized Kp1 are also reported. The reduced Fe proteins (Av2, Kp2) have similar CD and MCD, again indicating significant conservation of chromophore environment. The spectra clearly demonstrate the presence of a reduced bacterial ferredoxin-like (C
3-
) 4-Fe cluster. No obvious evidence of additional chromophores is observed. CD, MCD, and absorption spectra of Av1-oxidized Av2 are reported. The absorption spectrum shows the expected shoulder near 390 nm. The CD and MCD are characteristic of a C
2-
4-Fe cluster; in particular, the diagnostic near-infrared MCD peak is observed at ≈8300 cm
-1
. The CD of Av2 oxidized in the presence and absence of MgATP are radically different, providing the first direct evidence for MgATP interaction with Fe protein in this oxidation state.
Mechanism for discrimination between viral and host mRNA in interferon-treated cells.
Authors: Nilsen T W , Baglioni C
Published: May 2006

In extracts of interferon-treated HeLa cells, RNA covalently linked to double-stranded RNA (dsRNA) is preferentially degraded compared with mRNA not linked in dsRNA. This was established by following the degradation of poly(A)-containing mRNA annealed with poly(U), of poly(C)-containing encephalomyocarditis virus RNA annealed with poly(I), and of the replicative intermediate of the virus isolated from infected cells. In extracts of interferon-treated cells, dsRNA promotes the synthesis of a series of oligonucleotides, designated (2'-5')oligo(A), which in turn activate an endonuclease. Several lines of evidence suggest that the (2'-5')oligo(A) polymerase/endonuclease system is involved in the preferential degradation of mRNA linked to dsRNA. Conditions that prevent synthesis of (2'-5')oligo(A) prevent this preferential degradation, whereas addition of (2'-5')oligo(A) or conditions that favor its synthesis result in degradation of mRNA both linked and not linked to dsRNA. These results are best explained by a localized activation of the endonuclease near the dsRNA region of our model substrates. We propose that in infected cells activation of the endonuclease takes place near the replicative intermediates of RNA viruses. The replicative intermediates of encephalomyocarditis virus promote synthesis of (2'-5')-oligo(A) in extracts of interferon-treated cells and are degraded to a 20S "core" resistant to digestion with RNase A. This mechanism may be responsible for discrimination between viral and cellular mRNA in interferon-treated cells.
Multistep regulatory system for activation of a cyclic AMP-independent eukaryotic initiation factor 2 kinase.
Authors: Henderson A B , Miller A H , Hardesty B
Published: May 2006

Three functionally related components that block peptide initiation have been identified in lysates of rabbit reticulocytes. The components function consecutively in a cascade type sequence of reactions to cause phosphorylation of eukaryotic peptide initiation factor 2 (eIF-2). The eIF-2 kinase activated as part of this sequence has been tentatively identified as the same protein kinase that is activated by heme deficiency as part of the hemin-controlled repressor (HCR) system. The first component in the sequence is heat stable and can be reversibly activated by heat or pressure. It activates a second, heat-labile, component that in turn directly or indirectly activates the hemin-controlled eIF-2 kinase. This heat-labile component appears to function through proteolysis. This reaction sequence is not detectably affected by heme or cyclic AMP and thus appears to provide an alternative mechanism, independent of heme, for activation of the cyclic AMP-independent eIF-2 kinase of the HCR system.
Cycloheximide inhibition of hormonal induction of alpha 2u-globulin mRNA.
Authors: Chen C L , Feigelson P
Published: May 2006

The induction of hepatic alpha 2u-globulin synthesis by glucocorticoids in isolated hepatocytes occurs via an increase in the level of its mRNA as measured by cell-free translation and by hydbridization to an alpha 2u-globulin cDNA probe. To explore whether induction of this mRNA is a direct or an indirect consequence of the interaction of the dexamethasone-receptor complex with the alpha 2u-globulin genome, the requirement for ongoing protein synthesis was examined. Concentrations of cycloheximide too low to prevent precursor incorporation into total poly(A)-containing RNA do prevent the hormonal induction of alpha 2u-globulin mRNA. Furthermore, incorporation of 3H-labeled amino acids into total protein was decreased by only 40-50%, and the appearance of the dexamethasone-induced glycosylated forms of alpha 2u-globulin was completely prevented in these cycloheximide-treated hepatocytes. The results suggest that the synthesis of a protein mediator(s) may be required for the induction of alpha 2u-globulin mRNA by glucocorticoids and that the steroid-receptor complex may not interact directly with the alpha 2u-globulin genome.