bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclic AMP-dependent protein kinase I: Cyclic nucleotide binding, structural changes, and release of the catalytic subunits
Authors: Smith Stephen B. , White Hillary D. , Siegel Jeffrey B. , Krebs Edwin G.
Published: May 2006

Type I cyclic AMP (cAMP)-dependent protein kinase is composed of a dimeric regulatory subunit (R
2
) and two catalytic subunits (C subunits). The R
2
dimer binds four cAMP molecules to release the two C subunits. To characterize the cAMP binding sites and elucidate their role in the release of the C subunits, the R
2
dimer has been studied by equilibrium methods. The cAMP titration of R
2
was monitored by endogenous tryptophan fluorescence, and the results suggest one class of binding sites. The titration plot is monotonic for saturation of four sites per R
2
. A similar titration monitored by near-UV circular dichroic changes exhibited profound changes in the region of the
1
L
b
tyrosine and
1
L
a
and
1
L
b
tryptophan transitions; a plot of these data also showed a linear monotonic response. Thus, the fluorescence and circular dichroic changes show that cAMP binding to R
2
induces a conformational or structural change. The one apparent class of binding sites implies that all binding sites are characterized by similar
K
d
values or by
K
d
values much less than the receptor concentration. The reactivity of the cysteine sulfhydryl groups with 5,5′-dithiobis(2-nitrobenzoic acid) showed that saturation with cAMP indirectly protects one sulfhydryl group per R monomer. Analysis of cAMP activation of the holoenzyme, detected by phosphotransferase assays, showed that saturation of both cAMP binding sites per R monomer is necessary to effect the release of the C subunit. By using a fluorescent analog of cAMP, 1,
N
6
-etheno-cyclic AMP (ε cAMP), the (ε cAMP)
4
·R
2
complex was titrated with C subunit, causing the release of ε cAMP. The titration showed that the release of ε cAMP was a positive cooperative process; its Hill plot had a slope of 2.6 and the
K
a1
and
K
an
values obtained by extrapolation were 2.1 × 10
7
M
-1
and 5.0 × 10
8
M
-1
, respectively. The calculated ΔΔ
G
for first and last site coupling was 1.9 kcal/mol (1 cal = 4.18 J) of holoenzyme.
Recovery of protein synthesis after heat shock: prior heat treatment affects the ability of cells to translate mRNA.
Authors: Petersen N S , Mitchell H K
Published: May 2006

A mild heat shock at 35 degrees C, which induces heat shock gene expression, greatly enhances survival and the recovery of protein synthesis in Drosophila cells after a higher temperature heat shock. The 35 degrees C treatment is also effective in preventing heat-induced developmental defects in pupae. We show here that the major larval mRNAs are present in approximately normal (25 degrees C) concentrations after a 40.1 degrees C heat shock whether or not the animals receive a pretreatment. This indicates that the pretreatment affects translation directly rather than messenger concentration. We also observe selective translation of heat shock messages and some 25 degrees C messages during recovery from heat shock.
Photoisomerized bilirubin in blood from infants receiving phototherapy.
Authors: Lamola A A , Blumberg W E , McClead R , Fanaroff A
Published: May 2006

A pigment different from (Z,Z)bilirubin-IX alpha was detected by fluorometric methods in blood specimens from newborn infants undergoing blue-light therapy for unconjugated hyperbilirubinemia; it was not detected in specimens from infants not under therapy. The phototherapy-associated pigment has fluorescence, solubility, and photochemical properties that are identical to those exhibited by what are thought to be configurational (Z leads to E) isomers of bilirubin. It is concluded that isomerized bilirubin in the blood of neonates under phototherapy can reach as high as 15% of the total.
Detection of a recombinant murine leukemia virus-related glycoprotein on virus-negative thymoma cells.
Authors: Fischinger P J , Thiel H J , Ihle J N , Lee J C , Elder J H
Published: May 2006

X-irradiation of outbred Swiss mice resulted in the development of virus-free thymomas. When put in culture, a lymphoblastic cell line (NIXT) expressed neither particles nor infectious virus but supported the growth of pure ecotropic murine leukemia viruses (MuLVs) without generating any envelope recombinant (RM) MuLV in more than 20 months of culture. These cells did not support the growth of RM-MuLVs and completely excluded the entry of all RM-MuLV pseudotypes of murine sarcoma virus, suggesting specific viral interference. Radioimmunocompetition and immunofluorescence assays with broadly reactive anti-MuLV-p30 and -gp70 antisera were negative. However, in immunofluorescence with antisera specifically reactive against RM-MuLV gp70, about 5-20% of the population of parental cells or their clones were positive. NIXT cells treated with this antiserum bound protein A and exhibited complement-dependent cytotoxicity as assessed by several assays. NIXT cells could partially absorb neutralizing antibody specific for RM-MuLVs. Based on radioimmunoprecipitation tests, NIXT cells bore, on the cell surface, a glycosylated protein (gp70) reactive with RM subgroup as well as some group-specific anti-gp70 antisera. The glycoprotein was also found free in the supernates of NIXT cells. Using affinity chromatography, we determined the peptide pattern of the gp70 from NIXT cells to determine its structural relationship to gp70s of other MuLVs. NIXT gp70 was found to be highly related to class III endogenous xenotropic gp70s but, in addition, had peptide characteristics of RM-gp70s. Apparently, NIXT cells code for an unusual gp70 protein in the absence of other MuLV expression. The possible role of this glycoprotein in leukemogenesis is discussed.
Auditory brainstem response in dolphins.
Authors: Ridgway S H , Bullock T H , Carder D A , Seeley R L , Woods D , Galambos R
Published: May 2006

We recorded the auditory brainstem response (ABR) in four dolphins (Tursiops truncatus and Delphinus delphis). The ABR evoked by clicks consists of seven waves within 10 msec; two waves often contain dual peaks. The main waves can be identified with those of humans and laboratory mammals; in spite of a much longer path, the latencies of the peaks are almost identical to those of the rat. The dolphin ABR waves increase in latency as the intensity of a sound decreases by only 4 microseconds/decibel(dB) (for clicks with peak power at 66 kHz) compared to 40 microseconds/dB in humans (for clicks in the sonic range). Low-frequency clicks (6-kHz peak power) show a latency increase about 3 times (12 microseconds/dB) as great. Although the dolphin brainstem tracks individual clicks to at least 600 per sec, the latency increases and amplitude decreases with increasing click rates. This effect varies among different waves of the ABR; it is around one-fifth the effect seen in man. The dolphin brain is specialized for handling brief, frequent clicks. A small latency difference is seen between clicks 180 degrees different in phase--i.e., with initial compression vs. initial rarefaction. The ABR can be used to test theories of dolphin sonar signal processing. Hearing thresholds can be evaluated rapidly. Cetaceans that have not been investigated can now be examined, including the great whales, a group for which data are now completely lacking.
Developmental regulation of myelin basic protein in dispersed cultures
Authors: Barbarese E. , Pfeiffer S. E.
Published: May 2006

The expression of myelin basic protein, a major component of the myelin membrane, was studied in the absence of myelin formation in a unique situation in which these two processes have been uncoupled. The oligodendrocytes that contained myelin basic protein were identified by immunofluorescence in primary dispersed cultures derived from 20-day-old fetal rat brain. Their number increased 20-fold between 15 and 34 days in culture. Morphologically identifiable myelin was never observed. The oligodendrocytes elaborated a complex network of processes and membranous sheets resembling “unfurled” myelin. Myelin basic protein appeared concomitantly in the perikaryon, processes, and membranous sheets of the oligodendrocytes and remained distributed in these compartments throughout the culture period. The oligodendrocytes synthesized the four forms of myelin basic protein found in rodent brain with molecular weights of 21,500, 18,500, 17,000, and 14,000 and modulated their expression with time in culture. The onset of rapid myelin basic protein accumulation, as measured by radioimmunoassay, took place after 25 days in culture. Myelin basic protein accumulated at the rate of 0.2 fmol per oligodendrocyte per day and reached a level of 300 pmol/mg of protein by 34 days. By 60 days, the amount of myelin basic protein had declined to 100 pmol/mg of protein, a level maintained up until at least 120 days. When the amount of myelin basic protein was correlated with the number of oligodendrocytes, it was estimated that each induced cell contained on average 1 fmol of this protein at the three time points (15, 28, and 34 days in culture) at which cells were counted. Our results indicate that the accumulation, modulation of the molecular forms, and insertion of myelin basic protein into the membrane can occur in the absence of myelin formation, but that continued metabolic stability, subcellular sequestration, and fine control of the relative proportions of the different forms of myelin basic protein may be dependent on myelin morphogenesis.
Photosensitized electron transport across lipid vesicle walls: Enhancement of quantum yield by ionophores and transmembrane potentials
Authors: Laane Colja , Ford William E. , Otvos John W. , Calvin Melvin
Published: May 2006

The photosensitized reduction of heptylviologen in the bulk aqueous phase of phosphatidylcholine vesicles containing EDTA inside and a membrane-bound tris(2,2′-bipyridine)ruthenium(2+) derivative is enhanced by a factor of 6.5 by the addition of valinomycin in the presence of K
+
. A 3-fold stimulation by gramicidin and carbonyl cyanide
m
-chlorophenylhydrazone is observed. The results suggest that, under these conditions, the rate of photoinduced electron transfer across vesicle walls in the absence of ion carriers is limited by cotransport of cations. The rate of electron transfer across vesicle walls could be influenced further by generating transmembrane potentials with K
+
gradients in the presence of valinomycin. When vesicles are made with transmembrane potentials, interior more negative, the quantum yield of heptylviologen reduction is doubled, and, conversely, when vesicles are made with transmembrane potentials, interior more positive, the quantum yield is decreased and approaches the value found in the absence of valinomycin.
Thyroid or glucocorticoid hormone induces pre-growth-hormone mRNA and its probable nuclear precursor in rat pituitary cells.
Authors: Dobner P R , Kawasaki E S , Yu L Y , Bancroft F C
Published: May 2006

Thyroid or glucocorticoid hormone increases the synthesis of growth hormone (GH) by clonal lines of rat pituitary tumor cells. To investigate whether these increases arise from increased accumulation of GH-specific RNA sequences in the cytoplasm and nuclei of these cells, we adapted two existing procedures so that a 32P-labeled hybrid plasmid containing a cDNA sequence could be used to quantitate relative concentrations of the corresponding mRNA. One method (RNA gel blot hybridization) used electrophoresis of RNA, transfer to nitrocellulose paper, and hybridization to 32P-labeled plasmid. The other (RNA dot hybridization) used covalent attachment of RNA to activated cellulose paper squares and hybridization to 32P-labeled plasmid. As probe, we used a hybrid plasmid (pBR322-GH1) which we show by restriction analysis to contain a DNA sequence coding for rat GH. The results were comparable from both techniques and showed that incubation of GH3 cells with a thyroid hormone (triiodothyronine), a glucocorticoid hormone (dexamethasone), or both hormones caused an increase of cytoplasmic pre-GH mRNA sequences of about 4-, 22-, and 13-fold, respectively. Results obtained with the RNA gel blot hybridization method showed that hormonal stimulation leads to the induction of a single 1.0-kilobase species of pre-GH mRNA in the cytoplasm and of 2.7- and 1.0- kilobase species of GH-specific RNA in the nucleus.
Reactive site in human alpha 2-macroglobulin: circumstantial evidence for a thiolester.
Authors: Howard J B
Published: May 2006

The reaction of methylamine with alpha 2-macroglobulin (alpha 2M) results in the covalent modification of one glutamic residue per subunit as gamma-glutamylmethylamide [Swenson, R. & Howard, J. B. (1979) Proc. Natl. Acad. Sci. USA 76, 4313--4316]. Furthermore, alpha 2M can undergo specific peptide autolysis involving the same reactive glutamic residue [Howard, J. B., Vermeulen, M. & Swenson, R. (1980) J. Biol Chem. 255, 3820--3823]. During both reactions, a cysteinyl thiol is exposed and can be alkylated by iodoacetic acid. After alpha 2M was modified with [14C]methylamine and iodo[2-3H]acetic acid, a tryptic peptide was isolated that contained both labels in the same ratio as in the original protein. From the chymotryptic digest of the tryptic peptide, a single radiolabeled peptide was isolated. The amino acid sequence of the chymotryptic peptide was the same as that previously reported to include gamma-glutamylmethylamide. This is circumstantial evidence for a thiolester between the cysteine and a glutamic acid located three residues away in the primary sequence. A reaction mechanism involving a pyroglutamyl intermediate derived from the thiolester is suggested to explain the autolysis. Kinetic analysis of the autolysis reaction is consistent with this intermediate and mechanism.
Simulation of the initial stage of endochondral ossification: in vitro sequential culture of growth cartilage cells and bone marrow cells.
Authors: Suzuki F , Takase T , Takigawa M , Uchida A , Shimomura Y
Published: May 2006

Growth cartilage cells were isolated from the ribs of young rats and cultured at high cell density in Ham's F-12 medium supplemented with 10% fetal calf serum. During 7 days, glycosaminoglycans and proteoglycans were actively synthesized and secreted, forming a metachromatic matrix. When cultured together with growth cartilage cells precultured and biosynthetically prelabeled with 35SO4(2-) in their glycosaminoglycans, bone marrow cells caused release of 35S-labeled material into the culture medium. Glycosaminoglycan was also released by addition of conditioned medium obtained from cultures of bone marrow cells or peritoneal macrophages to the growth cartilage cell cultures. Electron microscopic studies of the extracellular matrix of growth cartilage cells cocultured with bone marrow cells showed that needles of apatite mineral were deposited within and in close apposition to the surfaces of matrix vesicles. These findings suggest that enzymes released from bone marrow cells or macrophages removed glycosaminoglycan or proteoglycans, which may be inhibitors of mineral growth, and consequently mineralization was initiated. From these findings, sequential culture of growth cartilage cells and bone marrow cells is promising as an experimental system for investigating the mechanism of the initial stage of endochondral ossification.
Secondary cytolytic T lymphocyte stimulation by purified H-2Kk in liposomes.
Authors: Herrmann S H , Mescher M F
Published: May 2006

Purified H-2Kk incorporated into lipid vesicles induced a secondary allogeneic cytolytic T lymphocyte response. However, the level of the response was much less than that generated by using purified plasma membranes containing an equivalent amount of antigen. Similarly, reconstituted membranes stimulated less effectively than did intact plasma membranes. In both cases the stimulating activity of the antigen was increased by including a detergent-insoluble membrane matrix fraction during formation of the liposomes or reconstructed vesicles. Liposomes formed in the presence of the matrix were larger, were more irregular in shape, and had a higher density than those formed in its absence. Both the H-2 antigen and matrix proteins were incorporated into the same vesicles. The greater antigenicity of H-2 in vesicles containing the matrix protein might be due to either the larger size of the liposomes or interaction of the antigen with a component(s) of the matrix.
13C NMR study of transamination during acetate utilization by Saccharomyces cerevisiae.
Authors: den Hollander J A , Behar K L , Shulman R G
Published: May 2006

13C NMR was used to follow the metabolism of [2- 13C]acetate and [1- 13C]acetate in aerobic suspensions of Saccharomyces cerevisiae. In the experiment with [2- 13C]acetate, the 13C label appeared first in glutamate C4 and subsequently in glutamate C2 and C3. After exhaustion of the acetate, the glutamate signals diminished and the aspartate C2 and C3 peaks increased. During a subsequent chase experiment with unlabeled acetate, the aspartate peaks decreased and the glutamate C2 and C3 peaks increased in intensity. These observations are interpreted in terms of an interplay between the glutamic-oxalacetic transaminase and Krebs cycle activity. This interpretation was confirmed by an experiment with the transaminase inhibitor 2-amino oxyacetate. During all of these experiments, we observed the formation of trehalose. The NMR gives a direct measurement of the label distribution and from that information it followed that the flows through the glyoxylate and the Krebs cycles are comparable. The intermediates citrate, succinate, fumarate, malate, phosphoenolpyruvate, 3-phosphoglycerate, and glucose 6-phosphate were identified in a 13C NMR spectrum of a perchloric acid extract taken during the metabolism of [2- 13C]acetate. Enrichment of the glutamate C5 position shows the existence of a futile cycle in which phosphoenolpyruvate, formed from oxaloacetate, returns to the Krebs cycle through pyruvate and acetyl CoA
Monoclonal antibodies to nucleic acid-containing cellular constituents: probes for molecular biology and autoimmune disease.
Authors: Lerner E A , Lerner M R , Janeway C A , Steitz J A
Published: May 2006

Mice of the strain MRL/Mp-lpr/lpr develop a lupus erythematosus-like syndrome that includes the production of autoantibodies specific for nucleic acid-containing cellular components. We have fused spleen cells from such a mouse with the myeloma SP 2/0 and examined the antibodies produced by the resultant cloned hybrid cell lines by using immunoprecipitation and immunofluorescence techniques. Three types of monoclonal antibodies, specific for Sm, DNA, or rRNA, all antigens to which patients who have lupus make antibodies, have been identified. Patient anti-Sm antibody had previously been reported to precipitate five small nuclear ribonucleoproteins that contain U-1, U-2, U-4, U-5, and U-6 RNAs. The monoclonal anti-Sm antibody gives the same immunoprecipitation pattern, providing direct evidence that the Sm antigen resides on all these RNA-protein complexes. Monoclonal anti-Sm antibody will be valuable in deciphering the biological function of these ubiquitous small nuclear RNPs. A simple competition radioimmunoassay using the monoclonal anti-Sm antibody to titer patient sera is also presented. Uses of monoclonal antibodies for the study of autoimmune disease are discussed.
Angiogenic activity from bovine retina: partial purification and characterization.
Authors: D'Amore P A , Glaser B M , Brunson S K , Fenselau A H
Published: May 2006

Bovine retinas contain a factor that stimulates proliferation of aortic endothelial cells in culture as well as neovascularization on the chicken chorioallantoic membrane. The stimulatory activity has been partially purified from a balanced salt solution extract of bovine retinas. The stability of the activity to acid pH was utilized as the first step in purification. The acid-treated material was subjected to ion-exchange chromatography on DEAE Bio-Gel A. The material that was eluted with 0.1 M NaCl/50 mM Tris.HCl (pH 7.6) stimulated both the proliferation of vascular endothelial cells in culture and angiogenesis in vivo. A molecular weight of between 50,000 and 100,000 for the native molecule is suggested by ultrafiltration and gel chromatography; gel electrophoresis of partially purified material under reducing and denaturing conditions revealed the presence of two major components with apparent molecular weights of 50,000 and 70,000. The endothelial cell stimulatory activity was stable to pH 4-9 and to heating at up to 60 degrees C for 30 min, to incubating for 1 hr with DNase or RNase, to incubating for 2 hr with immobilized Pronase or immobilized or soluble trypsin, and to treating with 1 mM 2-mercaptoethanol, 4 M urea, or 2 M guanidine. Heating at 65-75 degrees C for 30 min, boiling for 2 min, extreme acidic (pH 2) or basic (pH 12) conditions, treating with 0.02% NaDodSO4, or incubating (5 hr) with soluble Pronase destroyed the activity. The significance of an angiogenic factor derived from retina stems from the fact that neovascularization is a serious complication in a number of ocular diseases.
Simultaneous optical measurements of electrical activity from multiple sites on processes of cultured neurons.
Authors: Grinvald A , Ross W N , Farber I
Published: May 2006

Optical methods using changes in fluorescence and absorption of voltage-sensitive dyes were developed to record electrical activity from processes of nerve cells grown in monolayer culture. For transmission measurements, a merocyanine dye was discovered but was more sensitive than others previously tested on cultured neurons. Action potentials from the somata of these cells were detected without averaging, with a signal-to-noise ratio of 20:1. With this dye, electrical responses were simultaneously recorded from many points along the arborization of neuroblastoma cells by using a 10 X 10 array of photodiodes positioned in the microscope image plane. Frequently different processes had different shapes of electrical responses, suggesting regional specializations. Fluorescence measurements with an oxonol dye proved to be more sensitive than transmission measurements, particularly when recording from small processes. By changing the position of the cell relative to a laser microbeam while recording electrically from the cell body, it was possible to monitor the membrane potential in the cell body and in the process simultaneously. From the delay in response in the process, a lower limit for the mean conduction velocity of 0.2-0.6 m/sec was found for 2- to 6- micrometers processes. The mean space constants of processes were estimated by comparing the amplitudes of passive voltage responses in the cell body and growth cone. A lower limit of 400-950 micrometers was obtained for 4- to 7-micrometers processes.
Magnetic resonance spectroscopy of the primary state, P
F
, of bacterial photosynthesis
Authors: Bowman M. K. , Budil D. E. , Closs G. L. , Kostka A. G. , Wraight C. A. , Norris J. R.
Published: May 2006

We have obtained the magnetic resonance spectrum of the radical pair state P
F
by using reaction yield detected magnetic resonance spectroscopy. The magnetic resonance spectrum is quite sensitive to the local environment of P
F
. The data place limits on the lifetime of triplet P
F
and the distance of charge separation.
Translational control in lysates of Drosophila melanogaster cells.
Authors: Scott M P , Pardue M L
Published: May 2006

Cell-free protein-synthesizing systems made from Drosophila melanogaster cells were used to study the translational control induced in these cells by heat shock. Lysates of normally growing cells, termed 25 degrees C cells, translate both normal and heat shock mRNAs. Lysates of cells heat shocked at 36 degrees C for 1 hr, termed 36 degrees C cells, translate preferentially heat-shock mRNAs and a few 25 degrees C cell mRNAs. Thus, both lysates appear to reproduce the control displayed in vivo. Both lysates are optimally active at 28 degrees C, and all translations are done at that temperature, demonstrating that, once established, the discrimination system does not require heat-shock temperature for its activity. Addition of crude ribosome fractions from 25 degrees C cell lysates to lysates from heat-shocked cells "rescues" translation of 25 degrees C cell mRNA, which suggests that the discriminating elements are associated with ribosomes. Neither the heat-shock crude ribosome supplements nor the soluble fractions have any effect on either lysate. The experiments also show that RNA selection is determined by some feature of the RNA structure that is insensitive to protease digestion and phenol/chloroform extraction. The essential structural feature may not be unique to Drosophila mRNAs because the Drosophila lysate is capable of discriminating among mRNAs from other organisms.
Modification of microtubule steady-state dynamics by phosphorylation of the microtubule-associated proteins.
Authors: Jameson L , Caplow M
Published: May 2006

Phosphorylation of purified microtubule-associated proteins (MAPs) inhibits the rate and extent of MAP-stimulated microtubule assembly. The extent of microtubule assembly is reduced as a result of a decrease in the fraction of tubulin polymerized, without a significant change in the critical protein concentration. The decreased rate of microtubule assembly using phosphorylated MAPs reflects a reduction in microtubule nucleation resulting in fewer, but 2-fold longer, microtubules at steady state. Analysis of microtubule (MT) dynamics at steady state reveals that the rate of directional incorporation of subunits (flux) is 22 subunits.MT-1.sec-1 with phosphorylated MAPs, compared to 10 subunits.MT-1.sec-1 with unphosphorylated MAPs. The initial rate of disassembly determined by isothermal dilution is 232 subunits.MT-1.sec-1 for microtubules assembled with phosphorylated MAPs, compared to 102 subunits.MT-1.sec-1 for microtubules assembled with unphosphorylated MAPs. By using these results, the directionality (the number of successful subunit additions relative to the total number of association events per unit time) for subunit addition is found to be 0.1 for microtubules assembled with either phosphorylated or unphosphorylated MAPs. These observations are interpreted in terms of a mechanism in which phosphorylation of MAPs increases the rate of steady-state subunit flux by an equivalent enhancement of the rates of subunit association and dissociation, such that the critical protein concentration and directionality remain unchanged.
Proteinase inhibitor-inducing factor activity in tomato leaves resides in oligosaccharides enzymically released from cell walls
Authors: Bishop Paul D. , Makus Donald J. , Pearce Gregory , Ryan Clarence A.
Published: May 2006

The synthesis and accumulation of proteinase inhibitor I in excised tomato leaves can be induced with oligosaccharides obtained by fungal endo-α-1,4-polygalacturonase digestion of a pectic polysaccharide (
M
r
5000-10,000) isolated from tomato leaves. Active oligosaccharides were also released from isolated tomato leaf cell walls by endopolygalacturonases partially purified from tomato plants. It is suggested that oligosaccharides, released from plant cell wall pectic polysaccharides by either endogenous or exogenous endopolygalacturonases at a wound or infection site, may have hormone-like roles in regulating plant defense responses in unwounded tissues many centimeters away from the site of release.
ATP-dependent regulation of cytoplasmic microtubule disassembly.
Authors: Bershadsky A D , Gelfand V I
Published: May 2006

Indirect immunofluorescent staining with an antitubulin antibody was used for studying the role of ATP in the regulation of cytoplasmic microtubule disassembly. Depletion of the cellular ATP pool in cultured mouse fibroblasts with various inhibitors of energy metabolism leads to inhibition of the microtubule disassembly induced by colcemid or vinblastine. Glucose added to the inhibitor-containing incubation medium partially restores the cellular ATP content and abolishes the inhibition of microtubule disassembly. The metabolic inhibitors did not change [3H]colcemid uptake by the cells; therefore, their action on the microtubule disassembly was not caused by the reduction in intracellular colcemid. Addition of ATP to the cytoskeleton preparations obtained by Triton X-100 treatment of the cells markedly stimulates microtubule depolymerization. This effect was specific for ATP; it was not observed in the presence of GTP, UTP, CTP, ADP, AMP, adenosine 5'-(beta, gamma-methylene)triphosphate (a nonhydrolyzable analogue of ATP), or inorganic pyrophosphate or tripolyphosphate. Therefore, depletion of the cellular ATP pool reduces the rate of microtubule disassembly whereas addition of ATP increases it. These results suggest that a certain ATP-dependent reaction [most probably, phosphorylation of some of the microtubule protein(s)] controls microtubule disassembly in the cells.
Cell-cycle dependence of induced hemoglobin synthesis in Friend erythroleukemia cells temperature-sensitive for growth.
Authors: Conkie D , Harrison P R , Paul J
Published: May 2006

Friend erythroleukemia cells temperature-sensitive (ts) for growth have been used to study the cell-cycle dependence of erythroid differentiation. ts cells accumulated hemoglobin in response to inducer when replicating optimally at permissive temperatures. However, when arrested in the G1 phase of the cell cycle at a nonpermissive temperature, ts cells did not accumulate hemoglobin or globin mRNA nor did they differentiate terminally. Under these conditions, the viability of ts cells treated with inducer at the nonpermissive temperature remained high (70% of that of cells induced at the permissive temperature) as judged by cloning experiments. Total RNA and protein synthesis of G1 cells treated with inducer was 44-48% of that of randomly proliferating induced cells, in contrast to thymidine incorporation into DNA which decreased to 2% of that of cells replicating at the permissive temperature. Furthermore, G1 ts cells treated with inducer accumulated hemoglobin when released from growth arrest at the permissive temperature in the presence (but not in the absence) of inducer. These results are consistent with a requirement for a cell cycle-dependent event prior to commitment to differentiation.
Location of the 18/28S ribosomal RNA genes in two Hawaiian Drosophila species by monoclonal immunological identification of RNA.DNA hybrids in situ.
Authors: Stuart W D , Bishop J G , Carson H L , Frank M B
Published: May 2006

Using both heterologous rabbit antisera and mouse monoclonal antibody to RNA.DNA hybrids, we have mapped the in situ hybridization locus of the 18/28S ribosomal RNA fraction to a single large band on polytene autosome 3 in Drosophila heteroneura and Drosophila silvestris. This portion of the chromosome is not physically connected with the nucleolus at the end of larval salivary gland development. In mature larvae, little or no hybridization with the material in the nucleolus can be detected. In younger larvae, hybridization of the ribosomal RNA probe to the nucleolus itself can be observed. The chromosome 3 locus is the only band in the polytene genome that shows variation in size and intensity of staining between populations and species. The interband chromosome regions that are immediately distal or proximal to the 18/28S rRNA locus have been involved in a disproportionately large number of natural inversion breaks observed in the euchromatic portion of the polytene chromosome. In 104 species of Hawaiian Drosophila in which chromosome 3 polytene sequences have been determined, 15 breaks occur in these two regions. On a random basis, only one such break is expected. We propose that this locus may be flanked by substantial heterochromatic blocks which are not represented in the salivary gland chromosome.
Sequence homologies in the 5' regions of four Drosophila heat-shock genes.
Authors: Holmgren R , Corces V , Morimoto R , Blackman R , Meselson M
Published: May 2006

We report nucleotide sequences of the regions surrounding the 5' ends of the genes for Drosophila melanogaster heat-shock proteins hsp83, hsp68, and hsp26, located at chromosome positions 63BC, 95D, and 67B, respectively. As in other eukaryotic genes, the sequence T-A-T-A-A-A-A-T occurs about 30 nucleotides upstream from the sites of mRNA initiation. Three additional sequence homologies and a dyad symmetry were noted at approximately corresponding locations in the three genes and in the gene for another heat-shock protein, hsp70. We also found an intron near the 5' end of the hsp83 gene, with sequences at its boundaries typical of splice sites found in other organisms.
Somatostatin receptors: identification and characterization in rat brain membranes.
Authors: Srikant C B , Patel Y C
Published: May 2006

We have identified and characterized specific receptors for tetradecapeptide somatostatin (SRIF; somatotropin release-inhibiting factor) in rat brain using [125I]Tyr11]SRIF as the radioligand. These receptors are present in membranes obtained from a subfraction of synaptosomes. Membranes derived from cerebral cortex bind SRIF with high affinity (Ka = 1.25 X 10(10) M-1) and have a maximum binding capacity (Bmax) of 0.155 X 10(-12) mol/mg. Neither opiates nor other neuropeptides appear to influence the binding of SRIF to brain membranes. Synthetic analogs with greater biological potency than SRIF--[D-Trp8]SRIF, [D-Cys14]SRIF, and [D-Trp8, D-Cys14]SRIF--bind to the receptors with greater avidity than SRIF, whereas inactive analogs [(2H)Ala3]SRIF and [Ala6]SRIF exhibit low binding. The ratio of receptor density to endogenous somatostatin is high in the cortex, thalamus, and striatum, low in the hypothalamus, and extremely low in the brain stem and cerebellum. Thus, SRIF receptors in the brain appear to be a distinct, new class of receptors with a regional distribution different from that of endogenous somatostatin.
Multiple amino acid substitutions between murine gamma 2a heavy chain Fc regions of Ig1a and Ig1b allotypic forms.
Authors: Dognin M J , Lauwereys M , Strosberg A D
Published: May 2006

The complete amino acid sequence of the murine gamma 2a heavy chain CBPC-101 Fc region of allotype Ig1b was determined by automated and manual Edman degradation procedures. Both chemical and enzymatic cleavages were used to obtain peptides which were purified by gel filtration followed by high-pressure liquid chromatography. The sequence was in good agreement with that predicted from the nucleotide sequence of a gamma 2a gene of allotype Ig1b except for two positions. Comparison with published data on MOPC 173 gamma 2a heavy chain of allotype Ig1a revealed 8 amino acid substitutions in the CH2 domain (7% differences) and 18 substitutions (27%) in the CH3 domain. Many of the observed interchanges occur at positions at which murine heavy chains of other classes also differ from the gamma 2a chains. Our data suggest that the divergence of the gamma 2a Ig1a gene found in BALB/c mice and of the gamma 2a Ig1b gene found in CB 20 mice must have occurred long ago and that the CH2 domain was much more conserved than the CH3 domain.
Attachment site(s) of retinal in bacteriorhodopsin.
Authors: Katre N V , Wolber P K , Stoeckenius W , Stroud R M
Published: May 2006

After chemical reduction of the retinylidene-lysine Schiff base linkage in bacteriorhodopsin, the retinyl residue is covalently attached to Lys-216 (with a possible minor fraction on Lys-172) or to both Lys-216(172) and Lys-40/41. The linkage site (up to 100% on Lys-216; up to 70% on Lys-40/41) depends on whether the sample is reduced in the light or dark, whether the sample is light or dark adapted, and on temperature. Absorbance and circular dichroism spectra indicate that the retinyl residue is in its original binding site after reduction in the light. Thus, the different attachment sites may reflect changes that occur during the photoreaction cycle or during light/dark adaptation, or the reduction of accidental physiologically irrelevant Schiff base linkages to lysines close to the normal linkage in the structure of bacteriorhodopsin. In either case, the retinal does not leave its binding site. This last point severely limits the possible arrangements of the amino acid sequence in the bacteriorhodopsin tertiary structure and clearly distinguishes two models that are consistent with all criteria.
Novel opiate binding sites selective for benzomorphan drugs.
Authors: Chang K J , Hazum E , Cuatrecasas P
Published: May 2006

The simultaneous addition of [D-Ala2, D-Leu5]-enkephalin and morphiceptin at concentrations at which 98% of enkephalin (delta) and morphine (mu) receptors are occupied only partially inhibits the binding of [3H]diprenorphine to rat brain membranes. These conditions, furthermore, do not affect the curves for displacement of [3H]diprenorphine binding by unlabeled diprenorphine. These data suggest that [3H]diprenorphine binds to a third subtype of opiate binding site, which has high affinity for diprenorphine but very low affinity for mu and delta agonists. The [3H]-diprenorphine binding observed in the presence of morphiceptin and [D-Ala2, D-Leu5]enkephalin exhibits high affinity for several benzomorphan drugs in the chemical family of 6,7-benzomorphan (e.g., cyclazocine, ethylketocyclazocine, SKF 10047, UM 1072, oxilorphan, etc). Because of its selectivity for most benzomorphan drugs, this putative receptor site is tentatively referred to as a benzomorphan binding site. Its regional distribution in rat brain is similar to that of morphine (mu) receptors but differs from that for enkephalin (delta) receptors. The content of benzomorphan binding sites in rat brain is only one-half to one-third that of morphine receptors. The relative affinities of various opioids to morphine enkephalin, and benzomorphan binding sites are also described.
Site-specific interaction of DNA gyrase with DNA.
Authors: Fisher L M , Mizuuchi K , O'Dea M H , Ohmori H , Gellert M
Published: May 2006

DNA gyrase, in the presence of the inhibitor oxolinic acid, can induce double-strand DNA breakage at specific sites. The sequences at several sites have been determined. In addition, the structure of complexes formed between DNA gyrase and restriction fragments containing an oxolinic acid-promoted cleavage site has been examined by DNase protection methods. DNA gyrase protects more than 120 base pairs of DNA against pancreatic DNase in a region surrounding the cleavage site. Protection is observed both in the presence and absence of oxolinic acid. Protected DNA flanking the cleavage site contains DNase I-sensitive sites spaced on the average 10 or 11 base pairs apart. This result supports the view that, in the DNA gyrase--DNA complex, the DNA is largely wrapped on the outside of the enzyme.
Genes VI, VII, and IX of phage M13 code for minor capsid proteins of the virion.
Authors: Simons G F , Konings R N , Schoenmakers J G
Published: May 2006

The minor capsid proteins C and D from phage M13 have been characterized by differential amino acid labeling and amino-terminal sequence analysis. We demonstrate that D protein (Mr 12,260) is the product of gene VI, whereas the C component is composed of the products of both gene VII (Mr 3580) and gene IX (Mr 3650). Our data further show that the proteins of genes VI, VII, and IX are not subject to proteolytic processing but are packaged into mature virions as their primary translational products. On the basis of incorporation of specific amino acids, the copy numbers of these proteins in M13 virions could be estimated relative to the number of A protein molecules. The M13 phage contains on the average 5 molecules of A protein, 5 molecules of VI protein and 3-4 molecules of both VII protein and IX protein. These copy numbers remained unchanged in M13 recombinant phages of up to two times the length of wild-type phages, a fact that indicates that these minor capsid proteins are located at either one or both ends of the phage filament.
Subunit structure of cell surface proteins: disulfide bonding in antigen receptors, Ly-2/3 antigens, and transferrin receptors of murine T and B lymphocytes.
Authors: Goding J W , Harris A W
Published: May 2006

The surface proteins of lymphocytes from spleen and thymus and several cultured lymphoid tumor lines were radioiodinated in situ, solubilized with Triton X-100, and examined for the presence of disulfide-bonded subunits by two-dimensional (intact, reduced) NaDodSO4/polyacrylamide gel electrophoresis. [Hynes, R. O. & Destree, A. (1977) Proc. Natl. Acad. Sci. USA 74, 2855-2859]. Few lymphocyte surface proteins were found to consist of disulfide-bonded subunits, and the most prominent of these could be identified. In normal B lymphocytes and B-lymphoma cells, IgD or IgM (or both) were the major disulfide-bonded proteins, and these were easily detectable, even without immunoprecipitation. In contrast, analysis of thymocytes and T-lymphoma cells did not reveal any protein resembling immunoglobulin in its chain structure. The major labeled thymocyte membrane protein consisting of disulfide-bonded subunits was identified as the Ly-2/3 antigen. It appeared to contain disulfide-bonded homodimers of Mr 35,000 (alpha 2) noncovalently associated with a second pair of homodimers of Mr 30,000 (beta 2). Peptide mapping showed these polypeptides to be homologous. A third disulfide-bonded homodimer, which was heterogeneous in apparent Mr, appeared to be part of the Ly-2/3 complex. All cultured T- and B-lymphoma lines examined were found to possess a major surface protein that appeared to be a disulfide-bonded homodimer of a polypeptide of Mr 95,000. This protein was identified as the receptor for transferrin. It is suggested that the presence of two or more subunits in cell surface receptors renders their ligand functionally bivalent, making ligand-induced receptor aggregation possible.
Thyroid hormone attenuates and augments hepatic gene expression at a pretranslational level.
Authors: Seelig S , Liaw C , Towle H C , Oppenheimer J H
Published: May 2006

We have attempted to ascertain the proportion of the rat hepatic genome that is under the selective influence of thyroid hormones and to describe the response patterns of individual mRNA sequences in the transition between hypothyroidism and euthyroidism and between euthyroidism and hyperthyroidism. Poly(A)+RNA was extracted from livers of thyroidectomized, intact, euthyroid rats and of thyroidectomized rats rendered euthyroid and hyperthyroid with daily doses of triiodothyronine. The extracted RNA was translated in a reticulocyte lysate system in the presence of [35S]methionine, and the products were analyzed by two-dimensional gel electrophoresis. Triiodothyronine attenuates as well as augments the expression of certain genes at a pretranslational level. This could represent either a direct or an indirect action of the hormone. Triiodothyronine influences approximately 8% of the 231 mRNA sequences visualized, stimulating activity in 11 and inhibiting activity in 7 sequences. Translational activity of at least one mRNA sequence decreased in both thyroidectomized and hyperthyroid animals, compared to euthyroid levels. The relationship of mRNA response to receptor occupancy varied with examples of linear and amplified responses and responses that were maximal at less than full nuclear occupancy.
Structure and heme environment of beef liver catalase at 2.5 A resolution.
Authors: Reid T J , Murthy M R , Sicignano A , Tanaka N , Musick W D , Rossmann M G
Published: May 2006

Most of the amino acid side chains of beef liver catalase were clearly identifiable in the 2.5 A resolution electron-density map, and the results are in good agreement with the sequence [Schroeder, W. A., Shelton, J. R., Shelton, J. B., Roberson, B. & Apell, G. (1969) Arch. Biochem. Biophys. 131, 653-655]. The tertiary structure of one subunit consists of a large antiparallel beta-pleated sheet domain with helical insertions, followed by a smaller domain containing four alpha-helices. The heme group is buried at least 20 A below the molecular surface and is accessible by a channel lined with hydrophobic residues. The proximal ligand is tyrosine-357, while histidine-74 and asparagine-147 re the important residues on the distal side of the heme. The inhibitor 3-amino-1,2,4-triazole, which has been shown to covalently bond to histidine-74, can be built into the heme cavity with its N(2) atom coordinated to the heme iron.
An electron microscopic method for localization of ribosomal proteins during transcription of ribosomal DNA: a method for studying protein assembly.
Authors: Chooi W Y , Leiby K R
Published: May 2006

We describe a method for the localization of ribosomal proteins on electron microscopic spreads of active rRNA genes. The method consists of raising antibodies against Drosophila melanogaster proteins and allowing these antibodies to react with lysates of D. melanogaster egg chambers. The locations of the bound IgGs in the active transcripts are detected with goat anti-rabbit IgGs that have been labeled with electron-dense polymethacrylate spheres. By statistical analysis we can generate a confidence interval for the initial point of protein assembly for a particular protein. The first point of protein assembly for S14 is located near the 5' end of the pre-18S rRNA. In contrast, the first point of protein assembly for L4 is at 0.38 unit from the initiation point (a unit being the length of a ribosomal transcription unit). The binding patterns of S14 and L4 are consistent with the 5' proximal and the 5' distal orientations of the pre-18S and the pre-28S rRNAs. The method described here provides an approach to the elucidation of the assembly of eukaryotic ribosomal proteins in vivo.
Highly conserved glucose-regulated protein in hamster and chicken cells: preliminary characterization of its cDNA clone.
Authors: Lee A S , Delegeane A , Scharff D
Published: May 2006

A temperature-sensitive mutant K12 derived from a Chinese hamster fibroblast has been shown to overproduce three specific proteins of Mr 94,000, 78,000, and 58,000 when incubated at the nonpermissive temperature (40.5 degrees C). We previously identified these proteins as glucose-regulated proteins similar to those observed in chicken embryo fibroblasts when the cells are starved of glucose. In this report, we show that the Mr 78,000 proteins isolated from the hamster K12 cell line and from chicken embryo fibroblasts have identical electrophoretic mobilities in two-dimensional isoelectric focusing gels and nearly identical peptide maps. However, these proteins are different from heat-shock proteins previously described for animal cells. We have constructed a library of cDNA clones by using the RNA extracted from the hamster K12 cells incubated at 40.5 degrees C. Clones that hybridize preferentially with cDNA made from RNA at 40.5 degrees C were selected. By using the hybrid-selection technique, followed by in vitro translation, a cDNA clone containing a 2550-nucleotide insert coding for the hamster Mr 78,000 protein has been identified.
Population genetic theory of kin selection: Multiple alleles at one locus
Authors: Uyenoyama Marcy K. , Feldman Marcus W. , Mueller Laurence D.
Published: May 2006

Exact population genetic models of one-locus sib-to-sib kin selection with an arbitrary number of alleles are studied. First, a natural additive scaling is established for the genotypic value associated with probabilities of performance of altruism. Two classes of polymorphic equilibria are possible, one corresponding to the usual one-locus viability equilibria and the other reflecting the kin-selection assumptions of the model. At both, the covariance between additive genotypic value and genotypic fitness vanish. Further, the sign of this covariance determines the fate of rare alleles introduced near the first class of equilibria. In addition, the covariance explains the differences between Hamilton's rule, which results from Hardy-Weinberg assumptions, and exact initial increase conditions.