bluuebunny/crossref_metadata_2025 huggingface dataset:
Successful treatment of autoimmune disease in (NZB/NZW)F1 female mice by using fractionated total lymphoid irradiation.
Authors: Slavin S
Published: May 2006

Adult female (NZB/NZW)F1 hybrid mice with documented autoimmune glomerulonephritis resembling systemic lupus erythematosus were treated with fractionated total lymphoid irradiation (TLI), a modification of the radiotherapeutic regimen currently used for the treatment of Hodgkin disease. After TLI, proteinuria subsided in all mice that had undergone radiotherapy, and the mean survival increased from 359 days in untreated controls to 545 days. It is suggested that TLI should be further investigated as a new approach for immunoregulation of autoimmune disorders.
Tetraethylthiuram disulfide (Antabuse) inhibits the human malaria parasite Plasmodium falciparum.
Authors: Scheibel L W , Adler A , Trager W
Published: May 2006

Plasmodium falciparum in culture grows optimally at 3% oxygen. Oxygen levels down to 0.5% still support growth, but anaerobic conditions do not. These findings, and the absence of the Krebs cycle in Plasmodium, suggested that in this organism oxygen may not function in electron transport but rather may act through metalloprotein oxygenases. Tetraethylthiuram disulfide (Antabuse, disulfiram) and its reduction product diethyldithiocarbamate inhibit many metalloprotein oxygenases and have a lipid/H2O partition coefficient and high binding constant for metal ions, favoring selective toxicity to the malaria parasite. These compounds exhibited active antimalarial effects in vitro in concentrations down to 0.1 microgram/ml, the lowest level tested. Tetraethylthiuram disulfide at a level as low as 1 microgram/ml inhibited parasite glycolysis with no effect on glycolysis of normal erythrocytes. Erythrocytes pretreated with this drug at 10 microgram/ml did not support growth of the parasite.
Enzyme therapy in Fabry disease: differential in vivo plasma clearance and metabolic effectiveness of plasma and splenic alpha-galactosidase A isozymes.
Authors: Desnick R J , Dean K J , Grabowski G , Bishop D F , Sweeley C C
Published: May 2006

A pilot trial of enzyme replacement with splenic and plasma alpha-galactosidase A (alpha-D-galactosidase; alpha-D-galactoside galactohydrolase, EC 3.2.1.22) isozymes was undertaken in two brothers with Fabry disease, an X-linked glycosphingolipid storage disease. Six unentrapped doses (2000 units/kg) of each isozyme were administered intravenously to the respective recipients during a 117-day period. The circulating half-life of the splenic isozyme was about 10 min, whereas that for the plasma isozyme was approximately 70 min. No immune response was detected by skin and immunodiffusion tests or by alterations in the maximal activity or clearance kinetics for either isozyme after successive administrations. After each dose of the splenic isozyme, the concentration of the accumulated circulating substrate, trihexosylceramide (globotriaosylceramide), decreased maximally (approximately 50% of initial values) in 15 min and returned to preinfusion levels by 2-3 hr. In marked contrast, injection of the plasma isozyme decreased the circulating substrate levels 50-70% by 2-6 hr; the concentrations gradually returned to preinfusion values by 36-72 hr.
Ultrasensitive enzymatic radioimmunoassay: application to detection of cholera toxin and rotavirus.
Authors: Harris C C , Yolken R H , Krokan H , Hsu I C
Published: May 2006

Rotavirus and enterotoxin-producing bacteria are major causes of diarrheal disease in humans. A method of rapid diagnosis, ultrasensitive enzymatic radioimmunoassay, has been developed to quantitatively detect cholera toxin and rotavirus. The method uses features of both enzyme-linked immunosorbent assay and radioimmunoassay; however, the sensitivity of the assay is 100- to 1000-fold more sensitive than the two parent assays. Ultrasensitive enzymatic radioimmunoassay should also be useful in measuring other biologically important agents such as drugs and hormones.
Comparison of cell-fusing activity of brain suspensions from patients with Creutzfeldt-Jakob disease and other degenerative neurological diseases.
Authors: Moreau-Dubois M C , Brown P , Gajdusek D C
Published: May 2006

In vitro cell-fusing activity of brain suspensions from 33 patients with transmissible cases of Creutzfeldt-Jakob disease (CJD) was compared to activity of brains from 26 patients with a variety of other degenerative neurological diseases, and with activity of brains from 25 patients without neurological disease. A significantly higher proportion of CJD brains (61%) was positive than other neurologically diseased brains (31-35%) or the brains without neurological disease (0-4%). Although not yet sufficiently specific to be useful as a diagnostic test for human CJD, the assay nevertheless opens a line of investigation into the pathophysiology of degenerative neurological diseases and could prove immediately useful in rapidly locating material of maximum interest in purification procedures for experimental spongiform encephalopathy virus.
Phosphorus in Connecticut lakes predicted by land use
Authors: Norvell W. A. , Frink C. R. , Hill D. E.
Published: May 2006

The concentration of phosphorus in parts per billion (ppb) in 33 Connecticut lakes was predicted from information on land use and simple models for concentration decreases in lakes. The best predictions were obtained from [Formula: see text] where
P
is the concentration of phosphorus in ppb,
Q
is the water load on the lake in meters per year,
D
is the water export from the entire watershed in meters per year, and
U, A
, and
W
are the fractions of urban, agricultural, and wooded land, respectively, in the watershed. The phosphorus export coefficients and SEM, estimated by least squares regression, were 170 ± 21 mg per m
2
/yr for urban, 54 ± 15 mg per m
2
/yr for agricultural, and 10 ± 3 mg per m
2
/yr for wooded land.
P
± (SEM) 6.9 ppb was predicted.
Rapid ATP assays in perfused mouse liver by 31P NMR.
Authors: McLaughlin A C , Takeda H , Chance B
Published: May 2006

31P NMR was used to continuously monitor ATP and inorganic phosphate levels in perfused mouse liver. Under "optimal" conditions, the time resolution of the technique was approximately 1 min. In the absence of any metabolic perturbations the ATP level remained constant for at least 2 hr and decreased by only approximately 20% in 18 hr. Both ATP and inorganic phosphate levels responded to alterations in the oxygen supply to the liver. The half-time for this response was approximately 1 min, and the response to short periods of hypoxia or ischemia was partially reversible. The addition of insulin caused only a minor decrease in the ATP level but significantly decreased the rate of response of ATP and phosphate levels to hypoxia and ischemia.
Covalent association of protein with replicative form DNA of parvovirus H-1.
Authors: Revie D , Tseng B Y , Grafstrom R H , Goulian M
Published: May 2006

The double-stranded replicative form (RF) DNA of the autonomous parvovirus H-1 can be isolated from infected cells in a covalent complex with protein. The protein is present on most or all of the RF DNA, including actively replicating molecules, and is associated with the 5'-terminal endonuclease Hae III fragments of both the viral and complementary strands of RF. The size of the protein is estimated to be 60,000-70,000 daltons from its effect on buoyant density of DNA. DNA with covalently bound protein has not been found in H-1 virions.
Anisotropic molecular motion on cell surfaces.
Authors: Smith B A , Clark W R , McConnell H M
Published: May 2006

Photobleaching in a two-dimensional periodic pattern has been used to characterize the motion of succinylconcanavalin A receptors on the surfaces of mouse fibroblasts. These receptors were found to exhibit anisotropic two-dimensional diffusion in adherent cells having parallel stress fibers. Diffusion coefficients of the order of 10(-11) cm2/sec and 10(-12) cm2/sec were measured for diffusive motions parallel and perpendicular to these fibers. The determination of the anisotropic motion used a two-dimensional Fourier analysis. A study was made of the effect of changes in cell shape on the photobleach pattern.
Interaction of physalaemin, substance P, and eledoisin with specific membrane receptors on pancreatic acinar cells.
Authors: Jensen R T , Gardner J D
Published: May 2006

We have prepared 125I-labeled physalaemin and have examined the kinetics, stoichiometry, and chemical specificity with which the labeled peptide binds to dispersed acini from guinea pig pancreas. Binding of 125I-labeled physalaemin was saturable, temperature-dependent, and reversible and reflected interaction of the labeled peptide with a single class of binding sites on the plasma membrane of pancreatic acinar cells. Each acinar cell possessed approximately 500 binding sites, and binding of the tracer to these sites could be inhibited by physalaemin [concentration for half-maximal effect (Kd), 2 nM], substance P (Kd, 5 nM), or eledoisin (Kd, 300 nM) but not by cholecystokinin, caerulein, bombesin, litorin, gastrin, secretin, vasoactive intestinal peptide, glucagon, somatostatin, neurotensin, bovine pancreatic polypeptide, leucine-enkephalin, methionine-enkephalin, atropine, or carbamylcholine. With physalaemin, substance P, and eledoisin, there was a close correlation between the relative potency for inhibition of binding of labeled physalaemin and that for stimulation of amylase secretion. For a given peptide, however, a 3-fold higher concentration was required for half-maximal inhibition of binding than for half-maximal stimulation of amylase secretion, calcium outflux, or cyclic GMP accumulation. These results indicate that dispersed acini from guinea pig pancreas possess a single class of receptors that interact with physalaemin, substance P, and eledoisin and that occupation of 45% of these receptors will cause a maximal biological response.
Hormone receptor topology and dynamics: Morphological analysis using ferritin-labeled epidermal growth factor
Authors: McKanna James A. , Haigler Harry T. , Cohen Stanley
Published: May 2006

Previous studies using a biologically active 1:1 conjugate of EGF and ferritin (F-EGF) have traced the binding and internalization of the hormone molecules. In the present report, we develop ultrastructural criteria for identification of the F-EGF·receptor complex, and, thereby, enable utilization of the F-EGF as an indirect marker to localize the receptor for this peptide hormone. The ferritin cores of bound F-EGF are situated 4-6 nm from the extracellular surface of the membrane. When cells were incubated for up to 30 min at 37°C, this characteristic spatial relationship was observed in all uptake stages (surface clustering, endocytosis, and incorporation into multivesicular bodies), indicating that the hormone·receptor complex remains intact through these steps. However, when incubation was continued for periods sufficient to allow hormone degradation (30-60 min), pools of free ferritin were observed in lysosomes. In the presence of various amine inhibitors of hormone degradation, internalization and multivesicular body incorporation proceeded, but hormone·receptor degradation was blocked as evidenced by preservation of the ferritin—membrane relationship; i.e., no pools of free ferritin were seen after 60 min. These data provide morphological support for the hypothesis that down-regulation of surface receptors involves internalization of intact hormone·receptor complexes. In addition, we have developed a method for viewing the surface of intact cells
en face
, allowing closer scrutiny of the clustering of F-EGF·receptor complexes in the plane of the membrane prior to internalization. The particles in the F-EGF clusters observed by this method are spaced at 12 nm center-to-center, serving to set upper limits on the packing dimensions of the EGF·receptor complex.
Neoplastic transformation of epithelial cells in whole mammary gland in vitro.
Authors: Telang N T , Banerjee M R , Iyer A P , Kundu A B
Published: May 2006

Biological characteristics of nodule-like alveolar lesions (NLAL) induced by 7,12-dimethylbenz[a]anthracene (DMBA) in organ culture of whole mammary gland (BALB/c female mice) were assessed after transplantation into gland-free mammary fat pads of syngeneic virgin mice. (i) Tissue-fragment explants from NLAL areas of the gland produced abnormal lobuloalveolar (LA) outgrowths in 3 of 10 fat pads. (ii) Transplantation of dissociated cells of NLAL-derived LA outgrowths into 36 fat pads showed 100% LA outgrowths and 3 (8%) of these 36 outgrowths produced mammary carcinomas. (iii) The explants of dissociated cells from whole mammary glands treated with DMBA in culture produced full or partial LA structures in 2 of 56 outgrowths. (iv) The explants of dissociated cells prepared from outgrowths derived from outgrowths derived from explants as in iii produced 9 LA outgrowths in 16 instances; mammary tumor incidence in these outgrowths was 3 of 16 (18%). (v) The explants of tissue fragments from LA outgrowths as in iv produced LA outgrowths in 20 of 20 fat pads; mammary carcinomas appeared in 16 of 20 (80%) of these outgrowths. No NLAL was detectable in control glands treated with dimethyl sulfoxide (solvent for DMBA); explants of the control glands consistently produced ductal outgrowths and no tumor. This accomplishment of chemical carcinogen-induced neoplastic transformation of epithelial cells in vitro provides a model for studying carcinogenesis in an entire isolated organ.
Initiation of plasma prorenin activation by Hageman factor-dependent conversion of plasma prekallikrein to kallikrein.
Authors: Sealey J E , Atlas S A , Laragh J H , Silverberg M , Kaplan A P
Published: May 2006

Plasma prorenin is an inactive form of renin (EC 3.4.99.19) that can be converted to active renin in acid-treated plasma by an endogenous serine protease that is active at alkaline pH (alkaline phase activation). To identify this enzyme we first tested the ability of Hageman factor fragments, plasma kallikrein (EC 3.4.21.8), and plasmin (EC 3.4.21.7) to activate prorenin in acid-treated plasma. All three enzymes initiated prorenin activation; 50% activation was achieved with Hageman factor fragments at 1 microgram/ml, plasma kallikrein at 2-4 microgram/ml, or plasmin at 5-10 microgram/ml. We then showed that the alkaline phase of acid activation occurred normally in plasminogen-free plasma but was almost completely absent in plasmas deficient in either Hageman factor or prekallikrein; alkaline phase activation was restored to these latter plasmas when equal parts were mixed together. Therefore, both Hageman factor and prekallikrein were required for alkaline phase activation to occur. We then found that, although plasma kallikrein could activate prorenin in plasma deficient in either Hageman factor or prekallikrein, Hageman factor fragments were unable to activate prorenin in prekallikrein-deficient plasma. These studies demonstrate that alkaline phase prorenin activation is initiated by Hageman factor-dependent conversion of prekallikrein to kallikrein which, in turn, leads to activation of prorenin. In this fashion, we have revealed a possible link between the coagulation-kinin pathway and the renin-angiotensin system.
Role of thymidylate synthetase activity in development of methotrexate cytotoxicity.
Authors: Moran R G , Mulkins M , Heidelberger C
Published: May 2006

Methotrexate (MTX) inhibition of the growth of mouse or human leukemia cells in culture was partially prevented by either thymidine (dThd) or hypoxanthine. 5-Fluoro-2'-deoxyuridine (FdUrd) also decreased the growth-inhibitory potency of MTX in the presence of small concentrations of 5-formyltetrahydrofolate (citrovorum factor) and sufficient exogenous dThd to support the synthesis of thymidylate nucleotides by salvage mechanisms. In addition, citrovorum factor-induced reversal of MTX was several orders of magnitude more efficient in the presence of both FdUrd and dThd than in the presence of dThd alone or in the absence of both nucleosides. Likewise, the presence of FdUrd (3 microM) and dThd (5.6 microM) completely prevented the lethality of 0.3 mM MTX to L1210 cells in culture medium supplemented with micromolar concentrations of citrovorum factor. We propose that this protection against the cytotoxic effects of MTX by dThd, hypoxanthine, and FdUrd have a common biochemical mechanism--namely, inhibition of the de novo synthesis of thymidylate by either a direct [FdUrd; inhibition of thymidylate synthetase (thymidylate synthase; 5,10-methylenetetrahydrofolate:dUMP C-methyl-transferase, EC 2.1.1.45)] or indirect (dThd and hypoxanthine; feedback inhibition by anabolites on ribonucleotide reductase and deoxycytidylate deaminase) effect. The resultant decreased rate of loss of reduced folates due to de novo thymidylate synthesis would allow a higher degree of inhibition of dihydrofolate reductase to be endured without damage to the cell.
Higher molecular weight forms of immunoreactive somatostatin in mouse hypothalamic extracts: Evidence of processing
in vitro
Authors: Lauber Marc , Camier Maryse , Cohen Paul
Published: May 2006

Extracts of mouse hypothalamus made in acid/urea containing protease inhibitors were analyzed for somatostatin immunoreactivity after molecular sieve filtration on Sephadex G-50. Higher molecular weight (higher-
M
r
) somatostatin-like forms with apparent molecular weights of 15,000, 10,000, and 6000 could be identified, besides the molecular weight 1600 somatostatin. Immunological identities with somatostatin were unambiguously demonstrated by the analysis of the displacement curves in the radioimmunoassay. The
M
r
15,000, 6000, and 1600 species were purified by affinity chromatography on an anti-somatostatin immune serum covalent conjugate with Sepharose used as immunoadsorbant. After disulfide reduction by dithiothreitol, the size of the
M
r
15,000 and 6000 somatostatin-like species was assessed either by molecular sieve filtration or by polyacrylamide gel electrophoresis. The results indicated that the higher-
M
r
somatostatin-like species isolated from the hypothalamus did not result from hormone polymerization by means of disulfide interchange. The processing
in vitro
of the 15,000 higher-
M
r
form of somatostatin was achieved by proteolytic enzymes coeluted with this species during the fractionation of hypothalamic extracts. Under neutral pH conditions the intermediary higher-
M
r
forms were generated together with the
M
r
1600 somatostatin-like species. This processing activity could be either strongly inhibited at acidic pH or in acid/urea medium or else eliminated by selective immunoadsorption of the 15,000 higher-
M
r
form. Neither trypsin nor the γ subunit of 7S nerve growth factor was able to produce this processing, suggesting that enzymes with other kinds of specificity may be involved. It is concluded that somatostatin biosynthesis in the mouse hypothalamus may occur via a high-
M
r
precursor that is processed into intermediary forms leading to the tetradecapeptide hormone.
Diadenosine 5',5'''-P1,P4-tetraphosphate, a ligand of the 57-kilodalton subunit of DNA polymerase alpha.
Authors: Grummt F , Waltl G , Jantzen H M , Hamprecht K , Huebscher U , Kuenzle C C
Published: May 2006

By equilibrium dialysis a disadenosine 5',5'''-P1,P2-tetraphosphate (Ap4A) binding activity is shown to be present in mammalian cells. The Ap4A binding activity copurifies with DNA polymerase alpha during the isolation procedure, which includes chromatography on phospho-, DEAE-, and DNA-cellulose; gel filtration; sucrose gradient centrifugation; and electrophoresis in nondenaturing polyacrylamide gels. After these purification steps, DNA polymerase alpha appears to be homogeneous in nondenaturing polyacrylamide gels. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of such a purified DNA polymerase alpha preparation reveals seven distinct protein bands with apparent Mrs of 64,000, 63,000, 62,000, 60,000, 57,000, 55,000, and 52,000. By affinity labeling, the protein with Mr 57,000 has been shown to be the Ap4A-binding constituent of DNA polymerase alpha. The binding activity of DNA polymerase alpha for Ap4A is highly specific because neither structural analogs nor several other adenine nucleotides compete effectively with Ap4A for its binding site. The Ap4A binding site is lost in neuronal cells during maturation of rat brains concomitantly with the loss of DNA polymerase alpha and mitotic activity in those cells. From these results, DNA polymerase seems to be the intracellular target of Ap4A. This is discussed in respect to the recently reported of Ap4A to trigger DNA replication in quiescent mammalian cells [Grummt, F. (1978) Proc. Natl. Acad. Sci. USA 75, 371-375].
Alternative secondary structures of leader RNAs and the regulation of the trp, phe, his, thr, and leu operons.
Authors: Keller E B , Calvo J M
Published: May 2006

The trp, phe, his, thr, and leu operons of enteric bacteria are regulated by a transcriptional attenuation mechanism. Under conditions of amino acid sufficiency, transcription terminates at an attenuator site after a leader of about 150 nucleotides has been synthesized. Under conditions of limitation of a controlling amino acid, transcription continues past the attenuator into adjacent structural genes. As demonstrated by others, each of the five leader RNAs contains two regions of potential secondary structure which are partially overlapping. One of these regions occurs at the 3' terminus of the leader and is named the "terminator." The other region, which potentially can preclude the formation of the terminator, is named the "preemptor." Conditions that allow the preemptor to form result in derepression. We report here that the five published leader RNA sequences contain an additional potential region of secondary structure, which we call the "protector." The protector partially overlaps the preemptor in such a way that pairing of the former precludes pairing of the latter. For derepression to occur, a ribosome that is translating the leader must block the protector without blocking the preemptor, a condition that is met when the ribosome is arrested at the 3' end of a set of control codons. Including the protector in the model for attenuation explains why derepression of the operon does not result from the arrest of a ribosome at a codon preceding the control set. It also explains why termination is the outcome when transcription occurs in the absence of ribosomes. Finally, termination is the predicted outcome when unfettered translation of the leader RNA occurs, resulting in release of the ribosome at the translational stop signal.
Energetics of rapid transmembrane movement and of compositional asymmetry of phosphatidylethanolamine in membranes of Bacillus megaterium.
Authors: Langley K E , Kennedy E P
Published: May 2006

The energy requirements for the rapid transmembrane movement of phosphatidylethanolamine in membranes of Bacillus megaterium KM have been investigated by means of pulse label experiments. The transmembrane movement continues at a high rate in cells blocked in the production of metabolic energy by treatment with a combination of inhibitors. The movement is shown to be completely independent of the synthesis of lipid and of protein and, more generally, independent of sources of metabolic energy. The rate constant ki, defined as the fraction of the internal phosphatidylethanolamine that exchanges with the external layer of the membrane per unit time, has been found to have a value of about 0.1 per min. The compositional asymmetry of phosphatidylethanolamine in membranes of B. megaterium persisted, and indeed was somewhat enhance, in energy-poisoned cells under conditions in which rapid mixing of inner and outer layers was taking place. Therefore, the compositional asymmetry is not maintained by kinetic barriers to transbilayer exchange or by expenditure of metabolic energy. It must be an equilibrium condition, and presumably reflects the differential binding of phospholipids by proteins and other ligands on the two sides of the membrane.
Induction of erythroid differentiation of murine erythroleukemia cells by nicotinamide and related compounds.
Authors: Terada M , Fujiki H , Marks P A , Sugimura T
Published: May 2006

Nicotinamide and its analogues were evaluated for their activity as inducers of differentiation of murine erythroleukemia cells in culture. N'-Methylnicotinamide was the most effective of the compounds tested; at its optimal concentration it was more effective than dimethyl sulfoxide. With 8-10 mM N'-methylnicotinamide, almost all the cells contained hemoglobin (benzidine-reactive) after a 60-hr culture. Commitment to differentiate, assayed by transfer of the cells to semisolid medium without inducers, occurred much earlier and was more extensive with N'-methylnicotinamide than that with dimethyl sulfoxide or nicotinamide. Increase in globin mRNA was greater in the cells cultured with N'-methylnicotinamide than in cells cultured with dimethyl sulfoxide or nicotinamide. The relationship between the inducing activities of nicotinamide analogues and their effect on poly(ADP-ribose) polymerase in vitro was studied. All the compounds studied that had strong inhibitory effects on poly(ADP-ribose) polymerase in vitro induced differentiation of erythroleukemia cells in culture. This property is not a prerequisite of inducers; N'-methylnicotinamide did not inhibit the enzyme in vitro.
Cellular regulation of hemoglobin switching: evidence for inverse relationship between fetal hemoglobin synthesis and degree of maturity of human erythroid cells.
Authors: Papayannopoulou T , Kalmantis T , Stamatoyannopoulos G
Published: May 2006

To investigate whether the level of maturity of human erythroid cells influences the expression of the fetal hemoglobin program, we studied the relative production of fetal (Hb F) and adult (Hb A) hemoglobins during the maturation of erythroid clones produced in vitro by adult or neonatal erythroid stem cells. In both the adult and the neonatal cell cultures, clones composed of immature erythroblasts showed a significantly higher Hb F/Hb A ratio compared to the mature clones. Culture conditions enhancing erythroid cell maturity (such as an increase in the level of erythropoietin or culture time) decreased the relative synthesis of Hb F in the maturing erythroid cells. Direct immunofluorescence studies demonstrated earlier production of Hb F compared with Hb A during maturation of adult-origin HbF-synthesizing clones. The findings show that the final expression of Hb F is influenced by the degree of maturity of the terminally differentiated cells and suggest that, in addition to regulation at the level of erythroid stem cells, there is control of Hb F expression during erythroblast maturation. The inverse relationship between Hb F expression and level of cell maturity suggests that a regulatory mechanism operating throughout the process of erythroid stem cell differentiation/erythroblast maturation decreases the potential of Hb F expression as the development of the erythroid cell advances.
Human leukocyte interferon: production, purification to homogeneity, and initial characterization.
Authors: Rubinstein M , Rubinstein S , Familletti P C , Miller R S , Waldman A A , Pestka S
Published: May 2006

A method of fractionating proteins by high-performance liquid partition chromatography has been developed and used for isolation and purification to homogeneity of one of the species of human leukocyte interferon. The homogeneous interferon exhibited a sharp peak on high-performance liquid chromatography and a single narrow band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol. Extraction of the gel gave a single sharp peak of antiviral activity coinciding with the protein band. The specific activity of pure interferon was found to be 2--4 X 10(8) units/mg, based on amino acid analysis. The molecular weight is 17,500--18,000.
Membrane attack complex of complement: generation of high-affinity phospholipid binding sites by fusion of five hydrophilic plasma proteins.
Authors: Podack E R , Biesecker G , Müller-Eberhard H J
Published: May 2006

The molecular basis of the membranolytic activity of the membrane attack complex (MAC) of complement was investigated. By using density gradient equilibrium ultracentrifugation, the binding of egg yolk lecithin to the isolated MAC and to its intermediate complexes and precursor proteins was measured. No stable phospholipid--protein complexes were formed with the MAC precursor components C5b--6, C7, C8, and C9. Stable complexes of phospholipid and protein were formed by C5b--7, C5b--8, C5b--9, and the MAC (C5b--9 dimer) and they exhibited densities of 1.2164, 1.184, 1.2055, and 1.2275 g/ml, respectively. The molar phospholipid/protein ratios for the four complexes were determined to be: C5b--7, 399:1, C5b--5, 841:1; C5b--9, 918:1; and C5b--9 dimer, 1460:1. Electron microscopy of the isolated phospholipid--protein complexes revealed no lipid bilayer structures. The magnitude of the phospholipid binding capacity of the MAC is consistent with the interpretation that the MAC forms phospholipid--protein mixed in micelles in lipid bilayers and biological membranes and thus causes formation of hydrophilic lipid channels.
Presence of heparan sulfate in the glomerular basement membrane
Authors: Kanwar Yashpal S. , Farquhar Marilyn G.
Published: May 2006

The glomerular basement membrane was subjected to digestion with specific enzymes to determine the chemical nature (sialoglycoproteins, collagenous peptides, or glycosaminoglycans) of the anionic sites previously demonstrated in the laminae rarae. Enzyme digestion was carried out both
in situ
and
in vitro
. Kidneys were perfused
in situ
with enzyme solutions followed by perfusion with fixative containing the cationic dye, ruthenium red, to detect the anionic sites. Glomerular basement membranes were isolated by detergent treatment of glomeruli and incubated with enzyme solutions, followed by incubation with cationized ferritin (pI 7.3-7.5) to label the anionic sites. Only highly purified enzymes free of proteolytic activity were used. The findings were the same both
in situ
and
in vitro
. The anionic sites were unaffected by treatment with neuraminidase, chondroitinase ABC, and testicular or leech hyaluronidase. However, they could no longer be demonstrated after digestion with crude heparinase, purified heparitinase, or Pronase or after nitrous acid oxidation. The results demonstrate that the sites contain heparan sulfate since they are removed by treatment with heparitinase and by nitrous acid oxidation—procedures specific for heparan sulfate; and that sialoglycoproteins or other glycosaminoglycans do not represent major components of these sites since the latter are not affected by digestion with neuraminidase and other glycosaminoglycan-specific enzymes. Identical findings were obtained on basement membranes in other locations (Bowman's capsule, tubule epithelium, and endothelium of peritubular capillaries). The presence of heparan sulfate in the glomerular basement membrane is discussed in relation to the charge-selective properties of the glomerular filter and in relation to its potential involvement in various types of glomerular injury.
Amidolytic properties of single-chain activated Hageman factor.
Authors: Ratnoff O D , Saito H
Published: May 2006

Activation of Hageman factor (Factor XII) upon exposure to negatively charged agents has been attributed to proteolytic cleavage of this molecule. To examine this question, purified Hageman factor was exposed to Sephadex gels to which ellagic acid had been adsorbed. Such Hageman factor, separated from the gels and studied in the fluid phase, was amidolytic. Nonetheless, no cleavage of Hageman factor treated in this way could be demonstrated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Thus, activation of Hageman factor by negatively charged agents was not necessarily accompanied by molecular scission.
Myelin-associated glycoprotein demonstrated immunocytochemically in myelin and myelin-forming cells of developing rat.
Authors: Sternberger N H , Quarles R H , Itoyama Y , Webster H D
Published: May 2006

The unlabeled peroxidase-antiperoxidase method has been used with antiserum against "myelin-associated glycoprotein" to establish the presence of the glycoprotein in myelin and myelin-forming cells of the developing rat nervous system. Myelin-associated glycoprotein is found in oligodendroglial cytoplasm before the beginning of myelination. Staining intensity of oligodendroglia increases during early development and slowly declines during the period of rapid myelination. Myelin staining is confined to the periaxonal region of the myelin sheath and does not increase as large, compact sheaths are formed. Antiserum to central nervous system myelin-associated glycoprotein also stains Schwann cells in developing trigeminal ganglia and the periaxonal region of peripheral myelin sheaths.
Evidence for selection by male mating success in natural populations of Drosophila pseudoobscura.
Authors: Anderson W W , Levine L , Olvera O , Powell J R , de la Rosa M E , Salceda V M , Gaso M I , Guzmán J
Published: May 2006

Gene arrangement frequencies were determined at two stages in the life history of Drosophila pseudoobscura taken from nature. Three populations in the central highlands of Mexico were each sampled twice during 1976. Gene arrangement frequencies were measured in adult males and in larvae that were the offspring of females collected at the same time. The adult males were in all likelihood a representative sample of those who fathered the larvae produced by the wild females. Differences in gene arrangement frequency between these two life stages should indicate the operation of natural selection. One-third of our comparisons of common gene arrangement frequencies in males and in larvae from the next generation were statistically significant, as were one-third of our comparisons of total frequency arrays in the two life stages. We consider the components of selection that could produce such frequency changes and reason that male mating success must be the major one. Gene arrangement frequencies in the Mexican populations fluctuate within wide bounds. Selection must act to retain the polymorphism in the face of this flux in gene arrangement frequencies, and we suggest that male mating success plays an important role.
Termination of transcription by
Escherichia coli
RNA polymerase: Influence of secondary structure of RNA transcripts on ρ-independent and ρ-dependent termination
Authors: Adhya Samit , Sarkar Probir , Valenzuela David , Maitra Umadas
Published: May 2006

The effect of RNA secondary structure on ρ-independent and ρ-dependent termination of transcription of T3 DNA by
Escherichia coli
RNA polymerase has been studied by incorporating, into nascent transcripts, base analogs that lead to altered base-pairing properties. A guanine → hypoxanthine substitution, with attendant weakening of secondary structure, abolished the ρ-independent termination at 20% of the genome; in contrast, replacement of cytosine with 5-bromocytosine, which forms stronger pairs with guanine, enhanced termination at this site. ρ-Independent termination was not altered by replacing uracil with 5-bromouracil. There are two major ρ-dependent termination sites on the T3 DNA—at 8 and 15%. The termination activity of ρ in this system also depended on RNA secondary structure. The incorporation of 5-bromouracil instead of uracil into RNA did not alter the site specificity of ρ action but ρ was rendered inactive when cytosine was replaced by 5-bromocytosine. In contrast, replacement of GTP with ITP in the reaction increased ρ-dependent inhibition of RNA synthesis, caused production of heterogeneous-sized transcripts, and stimulated ρ-mediated ATP hydrolysis. The ρ-associated ATPase activity, in the presence of isolated T3 RNA, was also stimulated by inosine substitution. Furthermore, the temperature-sensitive ρ isolated from
rho
15 mutant of
E. coli
, which does not terminate transcription in the presence of the common rNTPs, was active when GTP was replaced with ITP. These results suggest that strongly paired G·C-rich regions in RNA stem-loop structures or RNA·DNA hybrids are essential for ρ-independent termination, whereas ρ-dependent termination requires weakly paired cytosine residues for its action.
Enzyme-catalyzed DNA unwinding: Studies on
Escherichia coli rep
protein
Authors: Yarranton Geoffrey T. , Gefter Malcolm L.
Published: May 2006

Replication
in vitro
of the replicative form (RF) I DNA of bacteriophage ϕX174 requires the phage-induced cistron
A
(cis
A
) protein, the host
rep
protein, DNA-binding protein, ATP, and DNA polymerase III plus replication factors. The
rep
protein is a single-stranded DNA-dependent ATPase. In this paper we show that ϕX174 RF I DNA cut by the cis
A
protein acts as a duplex DNA cofactor for the
rep
protein ATPase activity, provided that DNA-binding protein is present. In this latter reaction the duplex DNA is unwound by the
rep
protein with concomitant hydrolysis of ATP. The extents of ATP hydrolysis, DNA unwinding, and, where appropriate, DNA synthesis are proportional to the amounts of DNA-binding protein present. Two ATP molecules are hydrolyzed per base pair unwound. We propose that the obligatory requirement for the cis
A
protein in the unwinding of ϕX174 RF I DNA is not simply due to its endonuclease activity but rather is due to its provision of a site for the binding of the
rep
protein. The
rep
protein in the presence of DNA-binding protein, but in the absence of cis
A
protein, unwinds duplex DNA when one strand extends to generate a single-stranded leader region preceding the duplex. We show that
rep
protein translocates along the leader single strand in a 5′-to-3′ direction only and then invades the duplex DNA. The
rep
protein shows a directional specificity for translocation and unwinding. A model is presented to explain the mechanism of DNA unwinding catalyzed by the
rep
protein.
Complete amino acid sequence of a histidine-rich proteolytic fragment of human ceruloplasmin.
Authors: Kingston I B , Kingston B L , Putnam F W
Published: May 2006

The complete amino acid sequence has been determined for a fragment of human ceruloplasmin [ferroxidase; iron(II):oxygen oxidoreductase, EC 1.16.3.1]. The fragment (designated Cp F5) contains 159 amino acid residues and has a molecular weight of 18,650; it lacks carbohydrate, is rich in histidine, and contains one free cysteine that may be part of a copper-binding site. This fragment is present in most commercial preparations of ceruloplasmin, probably owing to proteolytic degradation, but can also be obtained by limited cleavage of single-chain ceruloplasmin with plasmin. Cp F5 probably is an intact domain attached to the COOH-terminal end of single-chain ceruloplasmin via a labile interdomain peptide bond. A model of the secondary structure predicted by empirical methods suggests that almost one-third of the amino acid residues are distributed in alpha helices, about a third in beta-sheet structure, and the remainder in beta turns and unidentified structures. Computer analysis of the amino acid sequence has not demonstrated a statistically significant relationship between this ceruloplasmin fragment and any other protein, but there is some evidence for an internal duplication.
Monovalent ionophores inhibit secretion of procollagen and fibronectin from cultured human fibroblasts
Authors: Uchida N. , Smilowitz H. , Tanzer M. L.
Published: May 2006

Procollagen and fibronectin are major products of confluent fibroblasts in culture and both are released from the cells. Procollagen is secreted by known pathways, while the mechanism of fibronectin release is controversial. We find that the secretion of both these proteins can be reduced to 20% by low concentrations (0.1-1 μM) of ionophores that have affinity for monovalent cations. In contrast, little effect upon secretion was found for similar concentrations of an ionophore that binds divalent cations. Electron microscopy showed that the inhibition of secretion is accompanied by accumulation of membranous vacuoles. We believe that the ionophores impede secretion by acting on the secretory structures rather than on the proteins themselves. Biochemical studies supported this interpretation because no changes were detected in hydroxylation or glycosylation of procollagen or glycosylation of fibronectin, nor were significant changes in cellular amino acid incorporation observed. Pulse-chase studies indicated that the rates of secretion were impaired by the ionophore without enhancing intracellular degradation. The decreased secretory rates accounted for the lower levels of procollagen and fibronectin in the culture medium; no evidence for increased catabolism of the secreted proteins was found. Secretion could be readily restored by removing the ionophore from the culture medium. The results indicate that procollagen and fibronectin may be simultaneously secreted, possibly utilizing a common pathway for secretion; the ionophores effectively interfere with cellular secretory pathways without impairing protein synthesis or protein glycosylation or altering protein catabolism.
Selection and characteristics of a Vibrio cholerae mutant lacking the A (ADP-ribosylating) portion of the cholera enterotoxin.
Authors: Honda T , Finkelstein R A
Published: May 2006

After mutagenesis with nitrosoguanidine and selection by immuno-halo techniques, an avirulent mutant, designated Texas Star-SR, which produces no detectable A (active; ADP-ribosylating) region of the cholera enterotoxin (choleragen) but produces the B region (choleragenoid) in amounts similar to the hypertoxinogenic wild-type parent Vibrio cholerae (biotype E1 Tor serotype Ogawa), has been isolated. The mutant retains the colonizing ability, motility, prototrophy, and serologic characteristics of the parent. In relevant intestinal experimental models, it has been shown to be avirulent and to induce protection against challenge with virulent cholera vibrios. The mutant appears to be suitable for further evaluation in volunteers as a candidate living enteric vaccine against cholera and related enterotoxic enteropathies.
Adenine nucleotide storage and secretion in platelets as studied by 31P nuclear magnetic resonance.
Authors: Ugurbil K , Holmsen H , Shulman R G
Published: May 2006

Suspensions of human and pig blood platelets have been studied by 31P NMR at 145.7 MHz and by chemical and radiochemical determination of nucleotide levels. In both types of platelets the cytoplasmic nucleotide pool, which was prelabeled by incubation with [14C]adenine, was selectively reduced by addition of H2O2/NaN3 or 2-deoxyglucose/antimycin A. After the reduction of cytoplasmic ATP in human platelets, the 31P NMR spectra showed an almost complete loss of the nucleoside di- and triphosphate resonances at temperatures examined (4--50 degrees C), indicating that only the cytoplasmic nucleotides had been observed, with no detectable contributions from the granular ATP, ADP, and pyrophosphate. Slow tumbling of the granular nucleotides, possibly due to aggregation, is the probable explanation of their undetectability at 145.7 MHz. Similar experiments showed that in pig platelets, granular ATP and ADP were not detected by 31P NMR at 4 degrees C but were observed at higher temperatures, indicating that aggregation may be occurring at the lower temperatures. Upon thrombin stimulation of human platelets, the NMR spectra and the chemical and radioactivity analyses showed that the granular adenylates and pyrophosphate were secreted, and that cytoplasmic ATP levels were appreciably reduced.
The rate of access to the organic ligand-binding region of serum albumin is entropy controlled.
Authors: Scheider W
Published: May 2006

The technique of real time dielectric relaxation measurement coupled with a conventional stopped-flow device has made it possible to measure the rates of association and dissociation of the complex of human serum albumin with its most prevalent ligands, the long-chain fatty acids. This association was previously shown to proceed in two steps: a fast, probably diffusion-controlled, nonspecific association, followed by a slower (approximately 3 sec--1) rearrangement of the intermediate protein--ligand configuration, whose kinetics is first order. By use of the Arrhenius relation and standard theory of rate processes it is determined that there is virtually no activation enthalpy in the forward binding reaction and that the rate of access to the interior hydrophobic binding region of serum albumin is controlled by a negative entropy of activation, reflecting a high degree of ordering in the transition state. A complete thermodynamic and kinetic profile of the association reaction is given.
Syndeins: the spectrin-binding protein(s) of the human erythrocyte membrane.
Authors: Yu J , Goodman S R
Published: May 2006

Two-dimensional tryptic and chymotryptic analyses of all the major bands in a sodium dodecyl sulfate/polyacrylamide gel of the human erythrocyte membrane show that each band has a characteristic map. However, band 2.1 (nomenclature of T. L. Steck) and several polypeptides below this band exhibit similar tryptic and chymotryptic peptide maps and thus appear to be a family of closely related proteins or degradation products. Furthermore, they all contain a subset of peptides that are accounted for by the peptides from two known spectrin-binding fragments. We show that both fragments derive from 2.1-related proteins and conclude that band 2.1 and its related proteins, which we name "syndeins", bind spectrin and connect it to the erythrocyte membrane.
Sodium-induced calcium release from mitochondria in brown adipose tissue.
Authors: Al-Shaikhaly M H , Nedergaard J , Cannon B
Published: May 2006

Coupled mitochondria of brown adipose tissue can accumulate Ca2+ if a substrate is present. The Ca2+ is released by addition of 20 mM Na+, but not by addition of K+ or choline +. Energy-dissipating Na+-induced Ca2+ cycling occurs maximally with 20 mM Na+ and 10 microM Ca2+. In brown adipocytes, the Ca2+ ionophore A23187 and the Na+ ionophore monensin increase respiration if substrate is added, and incubation in a low-Na+ buffer decreases norepinephrine-induced respiration. Thus Na+-induced Ca2+ release takes place in brown adipose tissue; released Ca2+ could have a regulatory or thermogenic role or both.
Biochemical basis for the enhanced toxicity of deoxyribonucleosides toward malignant human T cell lines.
Authors: Carson D A , Kaye J , Matsumoto S , Seegmiller J E , Thompson L
Published: May 2006

Human malignant T cell lines have high levels of deoxyribonucleoside phosphorylating activity and low levels of deoxyribonucleotide dephosphorylating activity. When incubated with deoxyadenosine or thymidine, the malignant T cell lines rapidly accumulate toxic concentrations of dATP and dTTP, respectively. This unusual pattern of deoxyribonucleotide metabolism renders the malignant T cells especially vulnerable to the toxic effects of deoxyribonucleosides and related analogues.
Biochemical basis for differential deoxyadenosine toxicity to T and B lymphoblasts: role for 5'-nucleotidase.
Authors: Wortmann R L , Mitchell B S , Edwards N L , Fox I H
Published: May 2006

Deoxyadenosine metabolism was investigated in cultured human cells to elucidate the biochemical basis for the sensitivity of T lymphoblasts and the resistance of B lymphoblasts to deoxyadenosine toxicity. T lymphoblasts have a 20-to 45-fold greater capacity to synthesize deoxyadenosine nucleotides than B lymphoblasts at deoxyadenosine concentrations of 50--300 micron. During the synthesis of dATP, T lymphoblasts accumulate large quantities of dADP, whereas B lymphoblasts do not accumulate dADP. Enzymes affecting deoxyadenosine nucleotide synthesis were assayed in these cells. No substantial differences were evident in activities of deoxyadenosine kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76) or deoxyadenylate kinase [ATP:(d)AMP phosphotransferase, EC 2.7.4.11]. The activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was increased 44-fold for AMP and 7-fold for dAMP in B lymphoblasts. A model for the regulation of deoxyadenosine nucleotide synthesis by 5'-nucleotidase activity is proposed on the basis of the observations.