bluuebunny/crossref_metadata_2025 huggingface dataset:
The molecular clock may be an episodic clock.
Authors: Gillespie J H
Published: May 2006

It is argued that the apparent constancy of the rate of molecular evolution may be an artifact due to the very slow rate of evolution of individual amino acids. A statistical analysis of protein evolution using a stationary point process as the null hypothesis leads to the conclusion that molecular evolution is episodic, with short bursts of rapid evolution followed by long periods of slow evolution. Such dynamics are incompatible with the neutral allele theory and require a revision of the standard interpretation of the molecular clock.
Characterization of heparan sulfate-proteoglycan of glomerular basement membranes.
Authors: Kanwar Y S , Veis A , Kimura J H , Jakubowski M L
Published: May 2006

Native and de novo synthesized heparan sulfate-proteoglycan (HS-PG) of basement membranes from isolated whole glomeruli were characterized. Sepharose CL-6B chromatograms of [35S]sulfate-labeled de novo synthesized HS-PG extracted from whole glomeruli indicated identical molecular weight characteristics to that isolated from purified basement membranes (Mr of intact HS-PG approximately equal to 130,000; Mr of chains approximately equal to 25,000). Electron microscopic autoradiography showed that almost all radioactive grains were localized to the basement membranes proper. The estimated Mr of core protein approximately equal to 18,000. The sedimentation coefficient of native intact HS-PG was 5.56 S, corresponding to a Mr between 150,000-250,000, a value in accord with gel filtration data on newly synthesized HS-PG. Physicochemical characteristics of HS-PG of native functional basement membranes differed remarkably from that isolated from the basement membrane-producing tumor, Engelbreth-Holm-Swarm sarcoma.
Murine monoclonal antibody to a single protein neutralizes the infectivity of human cytomegalovirus.
Authors: Rasmussen L E , Nelson R M , Kelsall D C , Merigan T C
Published: May 2006

Murine monoclonal antibodies to human cytomegalovirus (CMV) strain AD169 were selected that neutralized virus infectivity. One monoclonal antibody-producing hybridoma, 1G6, was used to produce ascites fluid from which immunoglobulin was isolated. This antibody efficiently neutralized CMV AD169, other laboratory strains (Towne, Davis), and clinical isolates of CMV in early tissue culture passage (less than 10) in the absence of complement. The antibody immunoprecipitated a single 86,000-dalton protein from both laboratory and clinical strains. This viral protein was demonstrated by indirect immunofluorescence to be localized in the cytoplasm of CMV-infected cells.
Identification of a 145,000 Mr membrane protein as the C3d receptor (CR2) of human B lymphocytes.
Authors: Weis J J , Tedder T F , Fearon D T
Published: May 2006

The C3d receptor (CR2) of human B lymphocytes mediates the binding to these cells of immune complexes that have activated the complement system and bear the fragments of C3, iC3b, C3d,g, and C3d. A 145,000 Mr membrane protein previously described as being recognized by the monoclonal antibody HB-5 and shown to be expressed only by B lymphocytes and B lymphoblastoid cell lines, such as Raji, was assessed for its possible identity as CR2. Treatment of Raji cells with HB-5 and goat F(ab')2 anti-mouse IgG (GaM) diminished the capacity of these cells to form rosettes with sheep erythrocyte (E) intermediates bearing 130,000 molecules of iC3b or C3d, whereas treatment with the monoclonal antibody alone had no effect. The capacity of peripheral blood B lymphocytes to bind EC3d was similarly inhibited by the combination of HB-5 and GaM. The possibility that HB-5 may interact with a site on CR2 that is distinct from the ligand binding site permitted the direct analysis of the capacity of the HB-5 antigen to bind to the C3 fragments. Protein A-containing Staphylococcus aureus particles to which HB-5 had been bound were incubated with detergent lysates of Raji cells and B lymphocytes under conditions that had been shown to be associated only with the binding of the 145,000 Mr antigen. These particles bearing HB-5 and antigen derived from either cell type were shown to adhere specifically to EiC3b and EC3d, demonstrating that transfer of the HB-5 antigen from CR2-bearing cells to S. aureus particles led to the acquisition of CR2 function by the particles. The additional findings that the relatively weak capacity of Raji cells to form rosettes with EC3b was inhibited by HB-5 and that the S. aureus particles bearing immunoadsorbed HB-5 antigen bound to EC3b indicated that the C3b-binding function of the CR1-negative Raji cell resides in CR2, rather than in other membrane proteins.
Differentiation of schistosomes by species, strain, and sex by using cloned DNA markers.
Authors: McCutchan T F , Simpson A J , Mullins J A , Sher A , Nash T E , Lewis F , Richards C
Published: May 2006

We have detected species, strain, and sex-specific genetic markers for the genus Schistosoma by Southern blot analysis of its DNA using cloned DNA segments of the Schistosoma mansoni ribosomal gene as probes. Restriction analysis of DNA from eight different strains of S. mansoni, from Africa and the Caribbean, revealed that the predominant or major DNA fragment containing the ribosomal gene unit was the same in each but that low copy number or minor fragments containing the gene varied. It was shown that the detection of these minor fragments could serve as the basis for both strain differentiation and the analysis of individual differences within a strain. Analysis of the parents and progeny of a genetic cross revealed sex-linked markers and suggested that these markers are inherited in a Mendelian fashion. DNAs from the species Schistosoma haematobium and Schistosoma japonicum were also analyzed. Differences in the length of the major repeating unit of the ribosomal gene served to distinguish each species. Furthermore, an array of minor bands was detected in each species, suggesting that strains of S. haematobium and S. japonicum could be differentiated in the same manner as S. mansoni strains.
Alteration of neural cell adhesion molecule (N-CAM) expression after neuronal cell transformation by Rous sarcoma virus.
Authors: Greenberg M E , Brackenbury R , Edelman G M
Published: May 2006

The effect of transformation by Rous sarcoma virus on the neural cell adhesion molecule N-CAM was assessed by immunoblotting, immunofluorescence staining, and an in vitro cell-cell aggregation assay using highly specific antibodies to the adhesion molecule. Expression of N-CAM was found to be temperature dependent in several rat cerebellar cell lines infected with a mutant Rous sarcoma virus that is temperature sensitive for transformation. At the nonpermissive temperature, these cells displayed significant quantities of N-CAM and aggregated rapidly by an N-CAM-mediated mechanism. However, when the cell lines were grown at the permissive temperature, they were morphologically transformed, contained much lower amounts of N-CAM, and aggregated poorly. A similar temperature dependence of N-CAM expression was not observed in cultured primary rat cerebellar cells nor in a chemically transformed neuronal cell line. In all of the cell lines, N-CAM occurred in the adult forms; the embryonic form has so far been observed in normal embryonic tissues and a few regions of the adult brain. The findings show that N-CAM prevalence at the cell surface can be modulated by transformation with clear-cut effects on cell-cell adhesion.
Functional molecular weight of the lac carrier protein from Escherichia coli as studied by radiation inactivation analysis.
Authors: Goldkorn T , Rimon G , Kempner E S , Kaback H R
Published: May 2006

Cytoplasmic membrane vesicles prepared from Escherichia coli containing multiple copies of the lac y gene were frozen in liquid nitrogen before or after generation of a proton electrochemical gradient (interior negative and alkaline) and irradiated with a high-energy electron beam at -135 degrees C. Subsequently, the lac carrier protein was extracted into octyl beta-D-glucopyranoside, reconstituted into proteoliposomes, and assayed for transport activity. Under all conditions tested, activity decreased as a single exponential function of radiation dosage, allowing straightforward application of target theory for determination of functional molecular mass. When lac carrier activity solubilized from nonenergized vesicles was assayed, the results obtained were consistent with a functional molecular size of 45-50 kDa, a value similar to the size of the protein as determined by other means. Similar values were obtained when the octyl beta-D-glucopyranoside extract was irradiated, and the target size observed for D-lactate dehydrogenase was in good agreement with the molecular size of this enzyme. Strikingly, when the same procedures were carried out with vesicles that were energized with appropriate electron donors prior to freezing and irradiation, a functional molecular size of 85-100 kDa was obtained for the lac carrier with no change in the target size of D-lactate dehydrogenase. In contrast, when the vesicles were energized under conditions in which the proton electrochemical gradient was collapsed, the target mass of the lac carrier returned to 45-50 kDa. The results indicate that the functional mass of the lac carrier protein is no greater than a dimer and suggest that the proton electrochemical gradient may cause an alteration in subunit interactions.
Proteolytic activation of a bioactive cardiac peptide by in vitro trypsin cleavage.
Authors: Currie M G , Geller D M , Cole B R , Needleman P
Published: May 2006

Mammalian cardiac atria possess several unidentified biologically active peptides. Fractionation of rat atrial extracts by gel filtration chromatography revealed two major fractions [apparent molecular weights of 20,000-30,000 (peak I) and less than 10,000 (peak II)], both of which were potent natriuretic agents (eliciting a 25-fold increase in sodium excretion) and smooth muscle relaxants. Vigorous treatment with trypsin (100 units/ml at 37 degrees C for 15 min) of both fractions abolished all biological activity. Further purification of the lower molecular weight fraction (peak II) by ion-exchange chromatography indicated two subfractions that possessed potent natriuretic activity and that preferentially relaxed either intestinal (designated peak IIA) or vascular (peak IIB) smooth muscle assay tissues. The similarity of the biological effect of the high (peak I) and low (peak II) molecular weight peptides led us to test the possibility of precursor-product relationship. Mild proteolytic treatment of the high molecular weight peptide with trypsin (1 unit/ml at room temperature) markedly enhanced the smooth muscle relaxant activity. Subsequent analysis of the trypsin (1 unit/ml)-treated high molecular weight peptide (peak I) by gel filtration and ion-exchange chromatography revealed that the peptide now resembled the low molecular weight peptides (peaks IIA and IIB) present in the original atrial extract. These data suggest that the cardiac atria contain a relatively inactive (smooth muscle relaxant) high molecular weight peptide and suggest that biologically active low molecular weight peptides can subsequently be generated by proteolytic cleavage.
Indefinite intertwining operators
Authors: Baldoni-Silva M. W. , Knapp A. W.
Published: May 2006

For a wide class of linear connected semisimple Lie groups, one obtains formulas limiting the Langlands parameters of irreducible unitary representations obtained from maximal parabolic subgroups. The formulas relate unitarity to the number of roots satisfying certain conditions. Some evidence is presented that the formulas are sharp. The results confirm aspects of conjectures that relate unitary parameters to cohomological induction.
Escape from strange repellers
Authors: Kadanoff Leo P. , Tang Chao
Published: May 2006

In a dynamical system described by a map, it may be that a “strange” sets of points is left invariant under the mapping. The set is a repeller if points placed in its neighborhood move away. An escape rate is defined to describe this motion. An alternative method of evaluating the escape rate, based on the consideration of repulsive cycles, is proposed. In the several cases examined numerically and analytically, the escape rate is shown to agree with the proposed formula.
Mechanism of antitumor drug action: poisoning of mammalian DNA topoisomerase II on DNA by 4'-(9-acridinylamino)-methanesulfon-m-anisidide.
Authors: Nelson E M , Tewey K M , Liu L F
Published: May 2006

The intercalative acridine derivative 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), but not its isomer o-AMSA, is a potent antitumor drug that in mammalian cells stimulates the formation of DNA strand breaks that are characterized by tightly bound proteins. Using purified mammalian DNA topoisomerases, we have analyzed the effects of these antitumor drugs on topoisomerase-DNA interactions. The antitumor drug m-AMSA dramatically stimulates the formation of a topoisomerase II-DNA complex that is detected on protein-denaturant treatment: both single- and double-stranded DNA breaks are produced and a topoisomerase II subunit is linked covalently to each 5' end of the broken DNA strands. The noncytotoxic isomer, o-AMSA, which does not induce significant amounts of DNA breaks in cultured cells, exhibits a correspondingly smaller effect in stimulating formation of the complex in vitro. The agreement between in vitro and in vivo studies suggests that mammalian DNA topoisomerase II may be the primary target of m-AMSA and that the drug-induced complex formation between topoisomerase II and DNA may be the cause of cytotoxicity and other effects such as DNA sequence rearrangements and sister-chromatid exchange.
Hepatocyte proliferation in vitro: its dependence on the use of serum-free hormonally defined medium and substrata of extracellular matrix.
Authors: Enat R , Jefferson D M , Ruiz-Opazo N , Gatmaitan Z , Leinwand L A , Reid L M
Published: May 2006

The culture conditions found to result in stable proliferation of normal rat hepatocytes are: (i) subconfluent cell densities; (ii) serum-free medium; (iii) hormonally defined medium containing epidermal growth factor, insulin, glucagon, prolactin, and other growth factors; and (iv) substrata of liver extracellular matrix depleted of growth inhibitors. Serum was found deleterious to parenchymal cells: it was inhibitory to the expression of liver-specific functions, cytostatic to parenchymal cells at all seeding densities, and cytotoxic to them at low seeding densities. These studies emphasize the relevance of synergies in the influences of hormones and extracellular matrix in regulating hepatocellular physiology.
Localization of RNA polymerase I in interphase cells and mitotic chromosomes by light and electron microscopic immunocytochemistry.
Authors: Scheer U , Rose K M
Published: May 2006

Rabbit antibodies to RNA polymerase I from a rat hepatoma have been used to localize the enzyme in a variety of cells at the light and electron microscopic level. In interphase cells the immunofluorescence pattern indicated that polymerase I is contained exclusively within the nucleolus. That this fluorescence, which appeared punctated rather than uniform, represented transcriptional complexes of RNA polymerase I and rRNA genes was suggested by the observation that it was enhanced in regenerating liver and in a hepatoma and was markedly diminished in cells treated with actinomycin D. Electron microscopic immunolocalization using gold-coupled second antibodies showed that transcribed rRNA genes are located in, and probably confined to, the fibrillar centers of the nucleolus. In contrast, the surrounding dense fibrillar component, previously thought to be the site of nascent pre-rRNA, did not contain detectable amounts of polymerase I. During mitosis, polymerase I molecules were detected by immunofluorescence microscopy at the chromosomal nucleolus organizer region, indicating that a considerable quantity of the enzyme remains bound to the rRNA genes. From this we conclude that rRNA genes loaded with polymerase I molecules are transmitted from one cell generation to the next one and that factors other than the polymerase itself are involved in the modulation of transcription of DNA containing rRNA genes during the cell cycle.
Time and voltage windows for reversing the electrical block to fertilization.
Authors: Shen S S , Steinhardt R A
Published: May 2006

The electrical block to fertilization of sea urchin eggs can be overcome by very brief periods of inside-negative egg membrane potential. Lytechinus pictus eggs whose membrane potentials have been clamped at +15 mV cannot be fertilized. If the membrane potential is repolarized to inside-negative voltages for a brief interval, the egg can be successfully fertilized. By varying the duration and voltage of these brief periods of inside negativity, we have uncovered three general properties of the electrically sensitive step in fertilization. First, a membrane-potential step that becomes rate limiting at inside-positive voltages can be initiated within a few milliseconds of inside negativity (30-60 msec at -60 mV). Second, at the time that the electrically sensitive step is being completed, there are other potential-independent steps with probably slower time constants because the duration of negativity was more effective applied as paired pulses rather than a single long pulse. Third, the permissive state is more quickly established by inside negativity than the nonpermissive state is established by inside positivity because the interval between paired pulses could be a few times longer than the effective single pulse in duration. In these voltage-clamped eggs the intervals from the successful completion of the electrically sensitive step to the next identifiable signs of activation were on the order of several seconds and highly variable.
Hormonal control of eclosion of flies from the puparium
Authors: Fraenkel Gottfried , Su Jack
Published: May 2006

Pharate flies (
Sarcophaga bullata
) very close to eclosion were injected with hemolymph from eclosing flies or various other preparations, kept at 10°C for 24 hr, returned to room temperature, and tested for eclosion behavior (ongoing, attempted, or completed eclosion) during the first 30 min, the subsequent 2½ hr, and after 51 hr. Flies injected with “eclosion” hemolymph showed significantly more eclosion activity during the first 30 min than noninjected controls, flies injected with hemolymph from larvae, or flies injected with Ringer solution.
groEL and dnaK genes of Escherichia coli are induced by UV irradiation and nalidixic acid in an htpR+-dependent fashion.
Authors: Krueger J H , Walker G C
Published: May 2006

Two proteins with molecular weights of 61,000 and 73,000 were found to be induced by UV light in Escherichia coli mutants in which the SOS responses are constitutively expressed. The induction of these proteins by UV light and nalidixic acid was shown to be independent of the recA+ lexA+ regulatory system. Analysis of these proteins by two-dimensional gel electrophoresis and comparison with the "heat-shock" proteins of E. coli revealed that the Mr 61,000 protein comigrated with the groEL gene product, that the Mr 73,000 protein comigrated with the dnaK gene product, and that other heat-shock proteins were also induced. The induction of groEL and dnaK by UV light and nalidixic acid is controlled by the htpR locus. The results suggest that the regulatory response of E. coli to agents such as UV light and nalidixic acid is more complex than previously thought.
Autoradiographic localization of angiotensin II receptors in rat brain.
Authors: Mendelsohn F A , Quirion R , Saavedra J M , Aguilera G , Catt K J
Published: May 2006

The 125I-labeled agonist analog [1-sarcosine]-angiotensin II ( [Sar1]AII) bound with high specificity and affinity (Ka = 2 X 10(9) M-1) to a single class of receptor sites in rat brain. This ligand was used to analyze the distribution of AII receptors in rat brain by in vitro autoradiography followed by computerized densitometry and color coding. A very high density of AII receptors was found in the subfornical organ, paraventricular and periventricular nuclei of the hypothalamus, nucleus of the tractus solitarius, and area postrema. A high concentration of receptors was found in the suprachiasmatic nucleus of the hypothalamus, lateral olfactory tracts, nuclei of the accessory and lateral olfactory tracts, triangular septal nucleus, subthalamic nucleus, locus coeruleus, and inferior olivary nuclei. Moderate receptor concentrations were found in the organum vasculosum of the lamina terminalis, median preoptic nucleus, medial habenular nucleus, lateral septum, ventroposterior thalamic nucleus, median eminence, medial geniculate nucleus, superior colliculus, subiculum, pre- and parasubiculum, and spinal trigeminal tract. Low concentrations of sites were seen in caudate-putamen, nucleus accumbens, amygdala, and gray matter of the spinal cord. These studies have demonstrated that AII receptors are distributed in a highly characteristic anatomical pattern in the brain. The high concentrations of AII receptors at numerous physiologically relevant sites are consistent with the emerging evidence for multiple roles of AII as a neuropeptide in the central nervous system.
Evidence that phosphorylation and dephosphorylation regulate the distribution of excitation energy between the two photosystems of photosynthesis
in vivo
: Photoacoustic and fluorimetric study of an intact leaf
Authors: Canaani Ora , Barber James , Malkin Shmuel
Published: May 2006

State 1-state 2 transitions in an intact tobacco leaf were monitored by the photoacoustic method. Modulated oxygen evolution yield and its enhancement by continuous far-red light (“Emerson enhancement”) were used to characterize the balance of light distribution between the two photosystems. These measurements were additionally supported by fluorimetry. Adaptation of the leaf to far-red light (λ [unk] 700 nm), mainly absorbed in photosystem I (light 1), results in state 1, where short-wavelength light (light 2) is distributed in favor of photosystem II. This is shown by a low yield of oxygen evolution, a high extent of Emerson enhancement, a concomitantly high extent of fluorescence quenching by far-red light, and a low ratio of the 77 K emission peaks at 730 and 695 nm. The magnitudes of these parameters were reversed when the leaf was adapted to light 2 (state 2), indicating a change towards a more equal distribution of the excitation between the two photosystems. Preincubation of an intact leaf with NaF, a specific phosphatase inhibitor, stimulated the extent of adaptation to light 2, shown by all the above criteria, and completely abolished adaptation to light 1. Light 1 preillumination prior to NaF treatment resulted initially in state 1, but then a transition to state 2 was irreversibly induced by any light. The NaF effect was specific because NaCl did not affect the state 1-state 2 transitions. Leaching out the NaF restored the original physiological transitions of the leaf. NaF presumably acts here in the same way as it acts in isolated thylakoids—by blocking the dephosphorylation of membranal proteins (particularly the chlorophyll a/b-protein complex) phosphorylated by a light 2-activated kinase. Our results give direct support to the suggestion [Allen, J. F., Bennett, J., Steinback, K. E. & Arntzen, C. J. (1981)
Nature
(
London
) 291, 25-29] that it is the phosphorylation level of thylakoid proteins that controls the light distribution between the two photosystems
in vivo
, shown previously in isolated thylakoids.
Cloned Drosophila alcohol dehydrogenase genes are correctly expressed after transfection into Drosophila cells in culture.
Authors: Benyajati C , Dray J F
Published: May 2006

We have obtained correct transcription of the cloned alcohol dehydrogenase (Adh) gene of Drosophila melanogaster after DNA-mediated gene transfer into Drosophila cells in culture. Supercoiled plasmids, each containing various regions of the Adh gene cloned in pBR327, were introduced into Schneider line 2 (SL2) cells by the calcium phosphate-DNA transfection technique. Although these cells do not normally express their endogenous Adh genes, they do express the exogenous genes as shown by primer extension and nuclease S1 analyses of RNA isolated 48 hr after transfection. The resulting alcohol dehydrogenase (ADH) transcripts, both the larval and adult types, have the correct 5' ends and are properly spliced. The transfected cells have also acquired ADH enzyme activity. The levels of enzyme activity and of ADH protein crossreacting material in cells transfected with different Adh plasmids correlate directly with the level of ADH transcripts. When a mutant Adh gene cloned from an ADH-negative mutant fly with a defect in the splicing of ADH RNA is transfected into the Schneider line 2 cells, the resulting ADH RNA is not spliced properly and there is no synthesis of ADH; thus, the mutant gene transfection into cell culture mimics the mutant phenotypes observed in the mutant fly.
Anti-hypervariable region antibody induced by a defined peptide: an approach for studying the structural correlates of idiotypes.
Authors: Chen P P , Houghten R A , Fong S , Rhodes G H , Gilbertson T A , Vaughan J H , Lerner R A , Carson D A
Published: May 2006

The structural correlates of idiotypes have been sought in several antibody systems. However, the precise molecular basis of idiotypes are exceedingly difficult to define. Antibodies of predetermined specificity can be induced by immunization with synthetic peptides. In the present experiments, a synthetic peptide corresponding to a hypervariable region on a monoclonal human IgM rheumatoid factor (Sie) has been used to induce specific anti-hypervariable region antibodies. The antibodies bound to the intact Ig molecule and to its isolated heavy chains but not to other IgM paraproteins nor to pooled human IgG. The binding of the antibody to the intact IgM was inhibited specifically by the free peptide, and the antibody activity was removed by a peptide-coupled affinity column. These results clearly demonstrate that specific anti-idiotypic antibody of predefined specificity can be induced by a hypervariable region peptide. Antibodies of this class may provide a new tool for defining the structural correlates of idiotypes.
Effects of food restriction on aging: separation of food intake and adiposity.
Authors: Harrison D E , Archer J R , Astle C M
Published: May 2006

Restricted feeding of rodents increases longevity, but its mechanism of action is not understood. We studied the effects of life-long food restriction in genetically obese and normal mice of the same inbred strain in order to distinguish whether the reduction in food intake or the reduction in adiposity (percentage of fatty tissue) was the critical component in retarding the aging process. This was possible because food-restricted obese (ob/ob) mice maintained a high degree of adiposity. In addition to determining longevities, changes with age were measured in collagen, immune responses, and renal function. Genetically obese female mice highly congenic with the C57BL/6J inbred strain had substantially reduced longevities and increased rates of aging in tail tendon collagen and thymus-dependent immune responses, but not in urine-concentrating abilities. When their weight was held in a normal range by feeding restricted amounts, longevities were extended almost 50%, although these food-restricted ob/ob mice still had high levels of adiposity, with fat composing about half of their body weights. Their maximum longevities exceeded those of normal C57BL/6J mice and were similar to longevities of equally food-restricted normal mice that were much leaner. Food restricted ob/ob mice had greatly retarded rates of collagen aging, but the rapid losses with age in splenic immune responses were not mitigated. Thus, the extension of life-span by food restriction was inversely related to food consumption and corresponded to the aging rate of collagen. These results suggest that aging is a combination of independent processes; they show that reduced food consumption, not reduced adiposity, is the important component in extending longevity of genetically obese mice.
Spiperone: evidence for uptake into secretory granules.
Authors: Dannies P S , Rudnick M S , Fishkes H , Rudnick G
Published: May 2006

Spiperone, a dopamine antagonist widely used as a specific ligand for dopamine and serotonin receptors, is actively accumulated into the F4C1 strain of rat pituitary tumor cells. The accumulation of 10 nM [3H]spiperone was linear for 3 min and reached a steady state after 10 min. Spiperone accumulation was reduced 50% by preincubation with 5 microM reserpine, an inhibitor of biogenic amine transport into secretory granules, and was also blocked by monensin and ammonium chloride, both of which increase the pH of intracellular storage organelles. Uptake was not affected by replacing sodium in the buffer with lithium at equimolar concentrations. Spiperone at 1 microM inhibited by over 50% serotonin transport into membrane vesicles isolated from platelet dense granules; this concentration inhibited the Na+-dependent plasma membrane transport system less than 10%. The data indicate spiperone specifically interacts with the secretory granule amine transport system and suggest that this transport system is found in the F4C1 pituitary cell strain as well as in platelets and neurons. The data also suggest that experiments utilizing spiperone to measure dopamine and serotonin receptors be interpreted with caution.
Strategy for the mass spectrometric verification and correction of the primary structures of proteins deduced from their DNA sequences.
Authors: Gibson B W , Biemann K
Published: May 2006

Fast atom bombardment mass spectrometry has been used to confirm and correct regions from the amino acid sequences of three large proteins, glutaminyl- and glycyl-tRNA synthetase from Escherichia coli and methionyl-tRNA synthetase from yeast, whose primary structures had been deduced from the base sequences of their corresponding genes. The strategy is based on a comparison of the molecular weights of the tryptic peptides predicted from all three reading frames of the gene sequences with those determined mass spectrometrically. The experimental molecular weights either match or differ and can be used to assess the correctness of the base sequences, identify errors that lead to frame shifts, premature stop codons, incorrect amino acids, etc., or identify the presence of posttranslational modifications. This method is very fast and requires little material (5-20 nmol).
Use of prokaryotic-derived probes to identify poly(sialic acid) in neonatal neuronal membranes.
Authors: Vimr E R , McCoy R D , Vollger H F , Wilkison N C , Troy F A
Published: May 2006

Three prokaryotic-derived probes to identify and study the temporal expression of polysialosyl units in neuronal tissue have been developed. A polyclonal antibody, a bacteriophage-derived endo-neuraminidase, and an Escherichia coli K1 sialyltransferase are all specific for either recognizing or synthesizing poly(sialic acid) containing alpha-2,8-ketosidic linkages. Polysialosyl immunoreactivity with apparent Mr values of 180,000-240,000 was specific for developing neuronal tissue; it was not detected in neonatal liver or kidney or in adult brain tissue. The developmentally regulated disappearance in poly(sialic acid) is consistent with the probes described here recognizing the polysialosyl carbohydrate units of a neuronal cell adhesion molecule (N-CAM). Treatment of brain extracts with a bacteriophage-derived endo-neuraminidase specific for alpha-2,8-linked polysialosyl units abolished the immunoreactivity. The material solubilized by endo-neuraminidase was isolated, reduced with borotritide, and shown to contain oligomers of sialic acid with three to six sialyl units. Treatment of the 3H-labeled oligosialic acid with exo-neuraminidase quantitatively converted the radioactivity to sialitol, establishing that the brain-derived oligomers were composed solely of sialic acid. A membranous sialytransferase from E. coli K1 that can transfer sialic acid to exogenous acceptors of oligo- or poly(sialic acid) also recognized rat brain membranes, further substantiating the presence of poly(sialic acid) in rat brain. This conclusion was confirmed by using a mutant of E. coli K1 that was defective in the synthesis of poly(sialic acid) and could only transfer sialic acid to exogenous acceptors of oligo- or poly(sialic acid). Sialyl polymer synthesis was restored in the mutant when brain membranes were added as exogenous acceptor.
Oxidative inactivation of glutamine synthetase subunits.
Authors: Nakamura K , Stadtman E R
Published: May 2006

Escherichia coli glutamine synthetase (GS) was inactivated by a nonenzymic mixed-function oxidation system composed of ascorbate, O2, and Fe(III). Partial inactivation of GS by this system leads to the formation of hybrid GS molecules (dodecamers) composed of both active and inactive subunits. Subunit interactions in these hybrid molecules are weaker than in the native enzyme, as indicated by the kinetics of subunit dissociation in the presence of 4 M urea. Heterologous subunit interactions in these hybrid molecules do not affect the affinity of active subunits for glutamate. Incubation of partially adenylylated GS preparations (n = 6.7) with the ascorbate system in the absence of substrates leads to preferential oxidative inactivation of unadenylylated subunits, whereas incubation in the presence of ATP and glutamate leads to preferential inactivation of adenylylated subunits.
Kinetics of electron transfer between cardiac cytochromes c1 and c.
Authors: Kim C H , Balny C , King T E
Published: May 2006

Highly purified cytochrome c1, which consists of only one heme peptide and does not form a stable c1-c complex (c1-H-c complex), was used in studies of electron transfer between cytochrome c1 and c. Results show that a stable and ionic-strength-sensitive c1-c complex (i.e., the c1-H-c complex) in the forms of the various oxidation states is not required, in contrast to the current belief of the participation of the complex in the electron transfer between cytochromes c1 and c. A minimum mechanism for electron transfer between these two cytochromes is suggested in accord with the experimental results.
Photoreduction of ferredoxin with various electron donors: Support for the Z scheme of photosynthetic electron transport
Authors: Neumann Joseph , Drechsler Ziwia
Published: May 2006

The currently accepted scheme for photosynthetic electron flow from water to ferredoxin (and subsequently to NADP), known as the Z scheme, envisions a linear electron flow that requires two photosystems joined by several electron carriers. The observation that the extent of photoreduction of ferredoxin depends on whether electrons are provided by water (a donor to photosystem II) or by artificial electron donors to photosystem I led Arnon
et al.
[Arnon, D. I., Tsujimoto, H. Y. & Tang, G. M.-S. (1980)
Proc. Natl. Acad. Sei. USA
77, 2676-2680] to question the validity of the Z scheme for photosynthetic electron transport. Our results show that this difference is not due to any inherent difference in electron transport but to the fact that when electron donors are added to chloroplasts they oxidize (in their oxidized state) reduced ferredoxin. Different electron donors oxidize reduced ferredoxin to a different extent; dehydroascorbate is a more potent oxidant than dithiothreitol. Dichlorophenol-indophenol is also a potent oxidant of reduced ferredoxin. The rate of NADP reduction and the
K
m
for NADP with various electron donors reflect the oxidative capacities of the electron donors and mediators. These results also explain the fact that NADP reduction with electron donors to photosystem I is less than that with water, despite the fact that electron flow from donor to artificial dyes can proceed at high rates.
Differential cellular distribution of cAMP-dependent protein kinase during development of
Dictyostelium discoideum
Authors: Schaller Kristin L. , Leichtling Ben H. , Majerfeld Irene H. , Woffendin Clive , Spitz Eva , Kakinuma Shizuko , Rickenberg Howard V.
Published: May 2006

It was shown previously by us that cAMP-dependent protein kinase activity in the cellular slime mold
Dictyostelium discoideum
increased during the early stages of development. Results from other laboratories showed that during the subsequent stage of cell differentiation and positioning, the accumulation of a number of prespore mRNAs and proteins (but not prestalk mRNAs and proteins) was dependent upon cAMP. The present communication describes the cellular distribution of the cAMP-dependent protein kinase at that stage of development. Pseudoplasmodia were disrupted, and prespore cells were separated from prestalk cells by sedimentation through a Percoll gradient. Prespore cells had approximately 4-5 times as much of both the catalytic and regulatory subunits of the cAMP-dependent protein kinase as did the prestalk cells. That the increase of cAMP-dependent protein kinase during development reflected
de novo
synthesis of the enzyme in both prespore and prestalk cells was demonstrated on the basis of [
3
H]leucine incorporation into the regulatory subunit. The findings are consistent with a role of the cAMP-dependent protein kinase in mediating the effects of cAMP on the synthesis of prespore-specific mRNAs and proteins at the stage at which cAMP appears to be required for the cell type-specific syntheses.
Cell-type-specific cDNA probes and the murine I region: the localization and orientation of Ad alpha.
Authors: Davis M M , Cohen D I , Nielsen E A , Steinmetz M , Paul W E , Hood L
Published: May 2006

Labeled cDNA probes highly enriched for B-cell-specific (B-T) and T-suppressor-cell-specific (Ts-B) sequences were used to screen a set of genomic cosmid clones spanning 230 kilobases of the murine immune response (I) region. With the B-cell derived probe, four I-region genes were detectable (A beta, E beta, E beta 2, and E alpha), as well as an additional (fifth) region of hybridization. The T-cell probe, prepared from a putative I-J positive suppressor cell hybridoma, was negative in a parallel experiment. A genomic fragment corresponding to the new region of hybridization seen with the B-cell cDNA probe identified a discrete mRNA species in RNA blotting analysis that had a pattern of expression strikingly similar to E alpha mRNA in a variety of lymphoid tumor lines. This fragment was also used to isolate cDNA clones from a library highly enriched (20-40 times) for B-cell-specific sequences (selected cDNA cloning). DNA sequence analysis of one of these cDNA clones indicates that this gene encodes the A alpha molecule of the d haplotype (Ad alpha). These findings establish that the order of the class II genes in the Ia complex is as follows: centromere-A beta-A alpha-E beta-E beta 2-E alpha.
Molecular cloning of cDNA for
Avena
phytochrome
Authors: Hershey Howard P. , Colbert James T. , Lissemore James L. , Barker Richard F. , Quail Peter H.
Published: May 2006

We have isolated several cDNA clones for phytochrome, a plant regulatory photoreceptor. A cDNA library was constructed by using etiolated
Avena
poly(A)
+
RNA enriched for phytochrome mRNA by size fractionation. Replicate arrays of colonies were differentially screened with cDNA probes made from poly(A)
+
RNA that had been either enriched in or depleted of phytochrome mRNA. Of the colonies hybridizing preferentially with the enriched probe, several contained plasmids that specifically selected phytochrome mRNA when assayed by hybridization-selection and translation. The largest such plasmid, pAP-2, was used to isolate clones from an
Avena
genomic library. One of these genomic clones was then used to screen a second cDNA library in an attempt to identify full-length phytochrome clones. The largest of the plasmids thus obtained, pAP-3, contains a 3.4-kilobasepair (kbp) insert, verified to contain phytochrome sequences by hybridization-selection and translation. Sequence analysis of pAP-2 and pAP-3 revealed that the two clones are identical in sequence through a 2.4-kbp region in which they overlap. However, the pAP-2 insert contains, in addition, 1.5 kbp of sequence of unknown origin, the apparent result of a recombination event. Blots of poly(A)
+
RNA hybridized with
32
P-labeled pAP-2 or pAP-3 show a single mRNA band at 4.2 kilobases. Blot analysis of RNA from dark-grown and from red-irradiated tissue demonstrates that a previously reported light-induced decrease in translatable phytochrome mRNA results from a decrease in physical abundance of this mRNA.
Comparison of the magnetic properties of deoxy- and photodissociated myoglobin.
Authors: Roder H , Berendzen J , Bowne S F , Frauenfelder H , Sauke T B , Shyamsunder E , Weissman M B
Published: May 2006

The magnetic susceptibility of photodissociated carbon monoxy myoglobin has been measured over the temperature range from 1.7 to 25 K at 10 and 50 kG with a superconducting susceptometer. The spin and the crystal field parameters of the iron ion were extracted by a spin Hamiltonian approach. Under equivalent conditions the magnetic susceptibility of deoxy myoglobin was measured. In both experiments the CO-bound protein was used as a diamagnetic reference. Above about 5 K the metastable photolysed state and the equilibrium deoxy form of myoglobin are magnetically indistinguishable and can be fitted with S = 2 and g = 2. The transition from spin 0 to spin 2 and the conformational changes known to accompany the electronic change thus also occur after photolysis at low temperature. At temperatures below 5 K, differences become apparent, indicating a somewhat smaller zero-field splitting in the photoproduct as compared to the ligand-free state at equilibrium. In qualitative agreement with observations made by other techniques, the data imply that even at 1.7 K substantial structural relaxation occurs in the heme region of myoglobin after photodissociation. The results are important for the interpretation of the ligand binding kinetics after flash photolysis at low temperature and contribute to the understanding of the relationship between electronic structure and function in heme proteins.
Down-regulation of the epidermal growth factor receptor in KB cells is due to receptor internalization and subsequent degradation in lysosomes.
Authors: Beguinot L , Lyall R M , Willingham M C , Pastan I
Published: May 2006

Using a monoclonal antibody to the human epidermal growth factor (EGF) receptor (EGF-R1), we have followed the metabolism of the receptor and the pathway of its internalization in KB cells after the addition of EGF. Measurement of surface binding of 125I-labeled EGF showed that about 80% of EGF binding activity disappeared from the plasma membrane after a 10-min exposure to EGF at 37 degrees C. Immunoprecipitation of the receptor from [35S]methionine-labeled cell extracts with EGF-R1 showed that EGF caused the receptor to be degraded with a half-life of 40 min. Immunofluorescence using EGF-R1 showed an EGF-dependent redistribution of the EGF receptor. In cells not exposed to EGF, almost all of the receptor was diffusely distributed on the cell surface. After EGF addition, the receptor was rapidly internalized, first appearing in small punctate organelles characteristic of receptosomes and then in larger perinuclear lysosome-like structures. By 120 min almost all of the immunoreactive EGF receptor had disappeared from the cells. Immunocytochemistry at the electron microscopic level confirmed these light microscopic findings. The diffusely distributed receptor on the cell surface first clustered into clathrin-coated pits in the presence of EGF, next was internalized into receptosomes, appeared transiently in transreticular Golgi elements, and finally was seen in lysosomes. This EGF-dependent down-regulation and degradation of the EGF receptor in KB cells provides a striking example of ligand-dependent clustering and internalization of a receptor, followed by degradation in lysosomes of both ligand and receptor.
Partial purification and characterization of the mRNA complementing a temperature-sensitive S-phase cell cycle mutation.
Authors: Fainsod A , Marcus M , Lin P F , Ruddle F H
Published: May 2006

E36ts24 is a temperature-sensitive (ts) cell cycle mutant derived from the Chinese hamster lung cell line E36. At the restrictive temperature for growth (40.3 degrees C), the mutant cells are arrested at early S phase. We have microinjected poly(A)+ RNA isolated from the wild-type E36 cells into the cytoplasm of E36ts24 cells arrested at early S phase after 24 hr of incubation at 40.3 degrees C. The ts mutation was transiently complemented in a significant fraction of the microinjected cells as evidenced by the incorporation of [3H]thymidine assayed by autoradiography. Microinjection of mRNA fractionated by methylmercuric hydroxide/agarose gel showed that the mRNA capable of transiently complementing the mutation in E36ts24 contains about 940 nucleotides. Hence, it can code at most for a protein containing about 230 amino acids. We estimate that the partial purification by fractionation of the mRNA active in the transient complementation is on the order of 100- to 200-fold.
Antibody against the insulin receptor causes disappearance of insulin receptors in 3T3-L1 cells: a possible explanation of antibody-induced insulin resistance.
Authors: Grunfeld C
Published: May 2006

The effect of a rabbit antibody induced against the rat insulin receptor (RAR) was tested using cultured 3T3-L1 fat cells. As previously seen with antibodies against the insulin receptor from patients with the type B syndrome of insulin resistance and acanthosis nigricans, RAR acutely mimicked the action of insulin by stimulating deoxyglucose uptake. After prolonged exposure of 3T3-L1 cells to RAR, insulinomimetic activity was lost and the cells became resistant to the action of insulin. This state of insulin resistance is similar to that seen with the human autoantibodies. However, unlike antibody from the patients, RAR did not acutely inhibit the binding of insulin to its receptor; rather, RAR increased the binding of insulin to its receptor by a mechanism consistent with an increase in the affinity of the receptor for insulin. With prolonged exposure to RAR there was a dramatic decrease in insulin-receptor binding on the treated 3T3-L1 fat cells. These results suggest that antibody against the insulin receptor induces insulin resistance by a mechanism that involves loss of cell-surface insulin receptors.