bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies of human myasthenia gravis: electrophysiological and ultrastructural evidence compatible with antibody attachment to acetylcholine receptor complex.
Authors: Rash J E , Albuquerque E X , Hudson C S , Mayer R F , Satterfield J R
Published: May 2006

Neuromuscular junctions from patients with early onset and chronic myasthenia gravis were examined by electrophysiological and ultrastructural techniques. Acetylcholine (AcCh) sensitivities were reduced by 34-63% in early onset myasthenia and 60-80% in chronic myasthenia. Ultrastructural analysis revealed that virtually all junctional folds of the early onset patients were intact but that the AcCh-receptor-rich crests of these folds were uniformly covered by an attached layer of 30 X 70 A particles arranged in small tufts or rosettes. In chronic myasthenic endplates, however, junctional fold crests were destroyed, apparently being replaced by vesicular membrane debris similarly labeled by tufts of 30 X 70 A particles. Thus, the initial reduction in junctional AcCh sensitivity observed in early onset myasthenia gravis may be attributed at least in part to in situ masking or inactivation of AcCh receptors, whereas the marked decrease in AcCh sensitivity observed in the chronic myasthenic patient may represent a combination of two factors: (a) in situ masking of AcCh receptors and (b) destruction of the receptor-containing crests of the junctional folds. These observations are compatible with an autoimmune etiology of myasthenia gravis initially involving an apparent antibody attachment to one or more components of the functional AcCh receptor complex, followed by systematic destruction and removal of junctional folds by both humoral and cell-mediated autoimmune responses.
Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers.
Authors: Axelrod D , Ravdin P , Koppel D E , Schlessinger J , Webb W W , Elson E L , Podleski T R
Published: May 2006

We have made direct, quantitative measurements of the lateral motion and age-dependent distribution of acetylcholine receptors (AChR) on the surface of rat myotubes in primary culture. AChR were fluorescently marked with tetramethylrhodamine-labeled alpha-bungarotoxin and AChR lateral motion was measured by the fluoresence photobleaching recovery technique. We found two coexisting distinct classes of AChR: (i) mobile, uniformly distributed AChR that appear on all myotubes shortly after fusion from myoblasts; and (ii) immobile, dense, highly granular AChR in patches of 10-60 mum size that appear shortly after fusion and disappear after myotubes have become extensively interconnected. In addition, evidence of turnover of AChR labeled with tetramethylrhodamine-alpha-bungarotoxin is seen in the gradual internalization of surface fluorescence within 36 hr after labeling. The relevance of these results to an understanding of the membrane dynamics and localization of muscle AChR is discussed.
Enzyme replacement therapy in Gaucher's disease: a rapid, high-yield method for purification of glucocerebrosidase.
Authors: Dale G L , Beutler E
Published: May 2006

Gaucher's disease is caused by a deficiency of the lysosomal enzyme glucocerebrosidase (glucosylceramidase; D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45); this disorder has been a leading candidate for enzyme replacement trials. A rapid, high-yield method for purification of glucocerebrosidase has been developed. Detergent extraction of human placenta was followed by salt fractionation, concanavalin A-Sepharose chromatography, organic solvent precipitation, and affinity chromatography on phosphatidylserine-agarose; the total yield is 60% with 6000-fold purification. Purified glucocerebrosidase has been administered intravenously to a volunteer Gaucher's patient on two separate occasions. For the first injection, the enzyme was entrapped in resealed erythrocytes; for the second injection, the enzyme was given without any carrier. The enzyme infusions caused no untoward effects.
Binding of Cibacron blue F3GA to proteins containing the dinucleotide fold.
Authors: Thompson S T , Stellwagen E
Published: May 2006

A simple, convenient, and sensitive spectrophotometric procedure is described for quantitative measurement of nucleoside phosphate binding sites constructed by the dinucleotide fold. The procedure involves difference spectral titration of such enzymes with the dye Cibacron blue F3GA in a spectral region remote from the intrinsic absorbance of proteins or natural ligands. The titration curves can be analyzed to determine the affinity of nucleoside phosphate binding sites for both the dye and the natural ligand over a potentially wide range of experimental conditions. The interaction of the dye with two proteins which contain the dinucleotide fold, lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) and phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3), is illustrated.
A gradient of adhesive specificity in developing avian retina.
Authors: Gottlieb D I , Rock K , Glaser L
Published: May 2006

Cell-cell adhesion was examined in the developing chick neural retina. A dorsoventral gradient of adhesive specificity in dissociated cells was detected which exhibits a complementarity such that the highest cell-cell affinities are exhibited between cells derived from the extremes of the gradient. If a nasotemporal gradient exists it must exhibit significantly lower cell-cell affinity. The relevance of these findings to pattern formation in the nervous system is discussed.
Properties of a toxin from the sea anemone Stoichacis helianthus, including specific binding to sphingomyelin.
Authors: Bernheimer A W , Avigad L S
Published: May 2006

Stoichactis helianthus toxin, a protein derived presumably from the nematocysts, was purified to homogeneity. It has a molecular weight of about 16,000, an isoelectric pH of 9.8, and it contains approximately 3.7% carbohydrate. It is powerfully hemolytic for erythrocytes derived from a variety of animal species, those of the cat being the most sensitive and those of the guinea pig the most resistant. The toxin is lytic also for rabbit blood platelets, and it destroys cultured fibroblasts but is inactive for several kinds of bacterial protoplasts and spheroplasts. The hemolytic activity is specifically inhibited by sphingomyelin, and it is proposed that this phospholipid is the constituent of the membrane which functions as receptor for the toxin. Supporting evidence includes the findings that enzymes known to destroy sphingomyelin (a) prevent erythrocyte membranes from inhibiting hemolysis, and (b) render erythrocytes resistant to lysis by the toxin. The mechanism underlying hemolysis may involve translocation of membrane sphingomyelin by virtue of a specific affinity of the coelenterate protein for this phospholipid.
A molecular concept of the properdin pathway.
Authors: Medicus R G , Schreiber R D , Götze O , Müller-Eberhard H J
Published: May 2006

The sequential events of the properdin system were analyzed. Properdin-depleted serum allows the formation of a Factor B- and D-dependent C3 convertase. This enzyme, called the properdin-receptor-forming enzyme, was shown to utilize a novel serum component, the initiating factor. The protein is a beta-globulin in precursor form and is distinct from immunoglobulins. The function of the enzyme is to deposit C3b on the surface of activator particles. Apparently doublets of C3b are required for the formation of the properdin-activating principle. It consists of a complex containing surface-bound C3b and activated Factor B. properdin precursor is activated by binding to this complex without detectable change in molecular weight. The transition of properdin precursor to activated properdin is probably caused by a conformational change. The complex, consisting of bound C3b, properdin, and activated Factor B, represents the enzyme that acts on C5, thereby initiating self-assembly of the membrane attack system. Native C3 is not needed for the function of the enzyme. It is disassembled by soluble C3 or C3b and its formation is under the control of the properdin-receptor-destroying enzyme, which may be identical with the C3b inactivator.
Monovalent derivatives of concanavalin A.
Authors: Fraser A R , Hemperly J J , Wang J L , Edelman G M
Published: May 2006

Monovalent dimers of concanavalin A (Con A) have been prepared by a combination of succinylation and photoaffinity labeling. Partial derivatization of native Con A using the photoaffinity label, p-azidophenyl-alpha-D-mannopyranoside, followed by affinity chromatography yielded a fraction that consisted of dimers with a single saccharide-binding site at pH 5. These monovalent dimers formed divalent tetramers at pH 7. In order to achieve a monovalent dimer at this pH, the divalent tetramers were succinylated by previously developed methods. Ultracentrifugation, equilibrium dialysis, and chromatographic experiments indicated that the resultant preparations consisted mainly of monovalent dimers which showed subunit exchange to yield about 15% divalent dimers after 12 hr at physiological pH. Freshly prepared material failed to agglutinate sheep erythrocytes at concentrations 500-fold higher than native tetravalent Con A. In addition, they showed saturating dose-response curves of mitogenic stimulation of mouse splenic lymphocytes. These curves resembled those of divalent succinyl-Con A but not those of the native molecule. Further development of methods for preparing stable monovalent derivatives of Con A should allow a refined analysis of the effects of lectin valence at the cell surface.
Inactivation of catalase monolayers by irradiation with 100 keV electrons.
Authors: Hahn M , Seredynski J , Baumeister W
Published: May 2006

A catalase monolayer adsorbed on a layer of arachidic acid deposited on a solid support was irradiated with 100 keV electrons simulating the conditions of electron microscopic imaging. Effective doses were calculated taking into account the angular and energy distribution of backscattered electrons. Enzymatic inactivation was chosen as the criterion for damage and was monitored by a rapid and quantifiable but nevertheless sensitive assay. Dose-response curves revealed that inactivation is a one-hit-multiple-target phenomenon, which is consistent with biochemical evidence for a cooperative function of subunits. The experimentally determined target size coincides fairly well with both calculated cross sections for inelastic interactions based on the atomic composition of catalase and with calculated cross sections for ionizing events based on the chemical bonds involved. This legitimates both types of calculations even for complex biomolecules.
The partial amino-acid sequence of an H-2K molecule.
Authors: Vitetta E S , Capra J D , Klapper D G , Klein J , Uhr J W
Published: May 2006

Eighteen of the NH2-terminal 27 amino acids of a murine H-2K molecule have been assigned. The approach used was to label murine splenocytes with a single radioactive amino acid, isolate the H-2K molecule by specific immunoprecipitation, electrophorese the dissolved precipitate on sodium dodecyl sulfate polyacrylamide gels, and subject the isolated H-2K peak to amino-acid analysis and automated sequencing.
Synthetic hexapeptide substrates and inhibitors of 3':5'-cyclic AMP-dependent protein kinase.
Authors: Kemp B E , Benjamini E , Krebs E G
Published: May 2006

The substrate specificity of the catalytic subunit of rabbit skeletal muscle 3': 5'-cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP: protein phosphotransferase) has been studied using the synthetic peptide Arg-Gly-Tyr-Ser-Leu-Gly corresponding to the sequence around serine 24, a phosphorylation site in reduced, carboxymethylated, maleylated (RCMM) chicken egg white lysozyme. This peptide served as a substrate for the enzyme and exhibited a 6-fold higher Vmax and a 100-fold higher Km than RCMM-lysozyme. Replacement of the arginine with glycine, histidine, or lysine resulted in a dramatic reduction in the Vmax. These results support the concept that arginine is an important residue in determining the substrate specificity of the protein kinase, predominantly influencing the Vmax of the phosphorylation reaction. Two synthetic peptides in which serine was replaced by an alanine acted as competitive inhibitors of phosphorylation of the synthetic peptide substrate and RCMM-lysozyme.
Deficiency in histone acetylation in nontransforming host range mutants of polyoma virus.
Authors: Schaffhausen B S , Benjamin T L
Published: May 2006

Histones H3 and H4 derived from transforming wild-type polyoma and simian virus 40 particles show extensive acetylation compared to the histones of the host cells. The same histone fractions derived from nontransforming host range mutants of polyoma virus fail to show this high degree of acetylation.
A 15-hydroxyprostaglandin dehydrogenase specific for prostaglandin A in rabbit kidney.
Authors: Oien H G , Ham E A , Zanetti M E , Ulm E H , Kuehl F A
Published: May 2006

Examination of a soluble fraction derived from homogenates of rabbit kidney papilla revealed the existence of a 15-hydroxyprostaglandin dehydrogenase specific for A-type prostaglandins. Prostaglandins of the E- and F-series were not substrates for this enzyme. In agreement with published data, the 15-hydroxyprostaglandin dehydrogenase(s) derived from the kidney cortex were found to degrade all prostaglandins examined (PGE, PGF, PGA) in the presence of added cofactor NAD. Thus it is evident that in this species the kidney 15-hydroxyprostaglandin dehydrogenases are anatomically compartmentalized so that the papilla is able to metabpable of degrading E-, F-, and A-type prostaglandins by this metabolic pathway.
Somatostatin conformation: evidence for a stable intramolecular structure from circular dichroism, diffusion, and sedimentation equilibrium.
Authors: Holladay L A , Puett D
Published: May 2006

Somatostatin is a hypothalamic tetradeca peptide that inhibits the release of growth hormone insulin, and glucagon. The circular dichroism spectrum is characterized by negative extrema at 238 nm and 270 nm, and a positive extremum at 225 nm. The far ultraviolet circular dichroism spectrum is consistent with the presence of ordered secondary structure such as beta-structure, but not alpha-helix. Sedimentation equilibrium results demonstrate that somatostatin exists in its monomeric form (i.e., a molecular weight of 1610 +/- 36 was obtained) and, thus, the structure must arise from intramolecular interactions. The diffusion constant of somatostatin was estimated to be 1.66 X 10(-6) cm2/sec. These data are consistent with an ellipsoidal rather than a spherical shape. The magnitude of the ellipticity at both 225 nm and 238 nm is quite dependent on guanidinium hydrochloride concentration; the midpoint occurs at about 3 M and the transition is cooperative-like. These data strongly suggest that somatostatin has a stable conformation in aqueous solution. A model, consistent with the results of the physicochemical studies and with semi-empirical rules for secondary structure formation, is proposed for somatostatin. The proposed structure consists of a hairpin loop with several residues in an antiparallel beta-pleated sheet, is somewhat elongated, and contains a hydrophobic domain at one end and a hydrophilic domain at the other end.
Antigen stimulation of prostaglandin synthesis and control of immune responses.
Authors: Webb D R , Osheroff P L
Published: May 2006

Within 2 min following the intravenous injection of sheep erythrocytes (sRBC) there occurs 20 to 80-fold increase in prostaglandin (PG) F2alpha in the spleen. This burst of synthesis is followed by a slow decline to control levels over the next 1-4 hr. No increase in splenic PGF2alpha levels is observed between 24 and 72 hr after injection. Injection of colloidal carbon results in a small increase, approximately 20% of the increase in PGF2alpha observed with sRBC. The early increase in splenic PGF2alpha levels stimulated by sRBC is also dependent upon thymus-derived (T) cells, since the increase is small or nonexistent in athymic mice and NZB mice. Also, the elevation of splenic PGF2alpha levels is blocked by the administration of indomethacin or Ro 20-5720, both of which block the synthesis of prostglandin. A small increase (2-fold) in PGF2alpha levels occurs in the thymus. A soluble antigen, bovine gamma globulin, stimulated a bimodal increase in splenic PGF2alpha levels, the early peak occurring at 2 hr and the later increase occurring at 48 hr. Using inhibitors of prostaglandin synthesis, it is possible to enhance the appearance of cells forming 19S antibody against sRBC, both in vivo and in vitro. Furthermore, inhibition of prostaglandin synthesis enhances DNA synthesis induced in a two-way mixed-lymphocyte reaction only in whole spleen cell cultures and not in cultures of spleen cells purified by passage over glass wool. Based on this evidence, it is proposed that the prostaglandins represent a major soluble mediator in the control of T cell-T cell interactions and also play an important part in T-B (bone-marrow derived) cell interactions.
Time-dependent response of soft polymers in moderately large deformations
Authors: Chang W. V. , Bloch R. , Tschoegl N. W.
Published: May 2006

We present a theory that successfully describes the time-dependent mechanical behavior of soft incompressible isotropic polymers in moderately large deformations. The theory is based on the introduction of a generalized measure of strain into the Boltzmann superposition integral.
Human pituitary growth hormone: restoration of full biological activity by noncovalent interaction of two fragments of the hormone.
Authors: Li C H , Bewley T A
Published: May 2006

The NH2-terminal 134 amino-acid fragment of the reduced-carbamidomethylated human somatropin molecule is found to react noncovalently with the COOH-terminal 51 amino-acid fragment in solutions of pH 8.4 at 2 degrees to restore full biological activity as evidenced by the rat tibia and pigeon crop-sac assays. In addition, circular dichroism spectra of the recombinant show the conformation to be completely repaired in comparison with that of the native hormone.
An assay of ribonuclease H, endoribonucleases, and phosphatases.
Authors: Starvianopoulos J G , Chargaff E
Published: May 2006

An assay for ribonuclease H (EC 3.1.4.34) is described which permits the estimation of the number and the type of cleavages produced by the enzyme when acting on the ribo moiety of a DNA-RNA hybrid substrate. With six different homopolymer hybrids tested, the number of enzymically induced breaks varied widely. The method is applicable to other endoribonucleases, phosphodiesterases, and phosphatases.
Regulation of guanylate and adenylate cyclase activities by lysolecithin.
Authors: Shier W T , Baldwin J H , Nilsen-Hamilton M , Hamilton R T , Thanassi N M
Published: May 2006

The guanylate cyclase activity [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] in membrane preparations from 3T3 mouse fibroblasts is stimulated approximately 5-fold by lysolecithin at concentrations of 100 mug/ml and above.
Hormonal control of meiosis: specificity of the 1-methyladenine receptors in starfish oocytes.
Authors: Dorée M , Guerrier P , Leonard N J
Published: May 2006

The specificity of the 1-methyladenine receptors, which are localized on the cell membrane of starfish oocytes, has been investigated in Marthasterias glacialis and Asterias rubens. Ability of N-1-substituted adenines to trigger the release of meiosis inhibition is conserved, or even increased, when the methyl group is replaced by larger substituent groups.
Cholera toxin interactions with thyrotropin receptors on thyroid plasma membranes.
Authors: Mullin B R , Aloj S M , Fishman P H , Lee G , Kohn L D , Brady R O
Published: May 2006

Unlabeled cholera toxin inhibits [125I]thyrotropin binding to thyrotropin receptors on thyroid plasma membranes. Maximal inhibition by cholera toxin does not exceed 40%, whereas unalbeled thyrotropin completely inhibits [125I]thyrotropin binding to these same membranes. Kinetic analyses of the binding data are compatible with the view that the cholera toxin decreases the number of receptor sites available to thyrotropin and that the mechanism by which the cholera toxin inhibits [125I]thyrotropin binding to these receptor sites involves both competitive and noncompetitive elements.
Search for antigens and antibodies crossreactive with type C viruses of the woolly monkeys and gibbon ape in animal models and in humans.
Authors: Stephenson J R , Aaronson S A
Published: May 2006

Several reports have indicated the presence of type-C viral antigens in human tumors and of viruses closely related to those of the woolly monkey and gibbon ape in cultured human cells. In the present studies, attempts to detect woolly monkeys viral antigens in human tissues, or antibodies directed against structural polypeptides of woolly monkey viruses in human sera, were unsuccessful, In contrast, it was possible to demonstrate viral antigens in tissues and antibodies reactive to viral components in several animal and even primate model systems. Further evidence against the presence of woolly monkey viruses in humans is our failure to identify spontaneous or chemically induced viruses of this group in more than 200 individual cultures of human origin examined. These findings argue against the likelihood that viruses closely related to the woolly monkeys virus are associated with human tumors or are common infectious agents of man.
Soluble dextran-hemoglobin complex as a potential blood substitute.
Authors: Tam S C , Blumenstein J , Wong J T
Published: May 2006

A complex between soluble dextran and human hemoglobin has been synthesized by two different methods. In the alkylation method, hemoglobin was allowed to react with bromoacetyl groups incorporated into the dextran; the yield of the complex was about 80% in terms of the hemoglobin used. In the dialdehyde method, hemoglobin was allowed to react with dialdehyde groups on the dextran generated by periodate oxidation; the yield of the complex was about 60%. Both soluble dextran-hemoglobin complexes could bind and release oxygen reversibly, but the oxygen-binding curves were shifted to the left relative to that of free hemoglobin. In the rabbit, the complex obtained by the alkylation method was excreted by the kidneys and cleared from the circulation much more slowly than free hemoglobin.
Markedly elevated angiotensin converting enzyme in lymph nodes containing non-necrotizing granulomas in sarcoidosis.
Authors: Silverstein E , Friedland J , Lyons H A , Gourin A
Published: May 2006

Sarcoidosis is a disease of unknown etiology that is characterized by the generalized formation of granulomas and is accompanied by elevation in the serum in less than half the patients of angiotensin converting enzyme, a dipeptidyl carboxypeptidase that catalyzes the conversion of the decapeptide, angiotensin I, to the pressor octapeptide, angiotensin II, and L-histidyl-L-leucine. Mean activity of angiotensin converting enzyme was elevated generally more than 10-fold in granuloma-containing lymph nodes, but not in lung in which normally it is abundant, in 19 of 20 patients with sarcoidosis. Angiotensin converting enzyme in lymph nodes from subjects with sarcoidosis was similar to the enzyme from normal lung and lymph node with respect to activity as a function of pH, inhibition of activity by EDTA and o-phenanthroline, gel filtration on Sephadex G-200, and requirement for chloride for activity, but appeared to be more heat labile. The data suggest that the granulomas in sarcoidosis may be the source of the elevated serum enzyme and that cells of the granulomas, particularly the epitheloid cells which appear by electron microscopy to have active protein biosynthesis, may be actively synthesizing the enzyme.
Retinal has a highly dipolar vertically excited singlet state: implications for vision.
Authors: Mathies R , Stryer L
Published: May 2006

We have measured the effect of an intense electric field on the absorption spectrum of solutions of all-trans retinal, its unprotonated Schiff base with n-butylamine, and the Cl- salt of this protonated Schiff base. The field-induced change in extinction coefficient as a function of wavelength was analyzed to determine the ground-state dipole moment (mug), the change in dipole moment on excitation (deltamu), and the direction of mug and deltamu). These experiments have shown that all three species become highly dipolar upon excitation to the first allowed excited singlet state (deltamu = 15.6, 9.9, 12D, respectively). The ground-state and excited-state dipole moments are nearly parallel to the long axis of these molecules. Excitation is accompanied by a shift of negative charge toward the carbonyl or Schiff base terminus, making the ionone end of these molecules positively charged. The large excited state dipole moment of all-trans retinal indicates that the vertically excited state, which is of 1Bu parentage (C2h), has become significantly mixed with even-parity states. On the basis of previous theoretical calculations, this mixing is expected to facilitate isomerization in the singlet manifold. We have also found that 11-cis retinal has a large deltamu (12.7 +/- 1.4 D) on excitation. In the visual pigments, the interaction of the excited-state dipole moment of retinal with a suitably located charged group could control the position of the absorption maximum. Also, the large shift in charge density upon excitation of retinal may lead to new electrostatic interactions between the chromophore and the protein that would act as a driving force for the initial conformational changes in visual excitation.
Maximal functions: Homogeneous curves
Authors: Stein Elias M.
Published: May 2006

Let
t
→ γ(
t
) be a homogeneous curve in R

n

. For suitable
f
, define [unk](
f
)(
x
) = sup

h
> 0

|(1/
h
) [unk]
0
h
f
(
x
- γ(
t
))
dt
|. Then ∥[unk](
f
)∥

p

≤

A
p

∥
f
∥

p

, 1 <
p
≤ ∞.
Defective thymine dimer excision by cell-free extracts of xeroderma pigmentosum cells.
Authors: Mortelmans K , Friedberg E C , Slor H , Thomas G , Cleaver J E
Published: May 2006

Crude extracts of normal human diploid fibroblasts and of human peripheral blood lymphocytes excise thymine dimers from purified ultraviolet-irradiated DNA, or from the DNA presumably present as chromatin in unfractionated cell-free preparations of cells that had been labeled with [3H]thymidine. Extracts of xeroderma pigmentosum cells from complementation groups A, C, and D also excise thymine dimers from purified DNA, but extracts of group A cells do not excise dimers from the DNA of radioactively labeled unfractionated cell-free preparations.
The endoplasmic reticulum: a cytochemist's view (a review).
Authors: Novikoff A B
Published: May 2006

Enzyme cytochemistry has been used, at the light and electron microscope levels, to "mark" cytoplasmic organelles of mammalian cells. Catalase cytochemistry permitted identification of microperoxisomes, apparently ubiquitous organelles that are attached by numerous slender connections to the endoplasmic reticulum. Thiamine pyrophosphatase and acid phosphatase cytochemistry can be used to distinguish between the Golgi apparatus and a specialized acid-phosphatase-rich region of smooth endoplasmic reticulum (ER) that appears to be involved in: (a) the formation of lysosomes and melanin granules: (b) the processing and packaging of secretory materials in endocrine and exocrine cells; and (c) the metabolism of lipid. The acronym GERL has been given to this region of smooth ER because it is located at the inner or "trans" aspect of the Golgi apparatus and because it appears to produce various types of Lysosomes.
Radioimmunoassay for intact Gross mouse leukemia virus.
Authors: Yalow R S , Gross L
Published: May 2006

A radioimmunoassay for intact Gross leukemia virus has been developed using 125I-labeled Gross virus grown in tissue culture and guinea pig antisera to Gross virus grown either in tissue culture or harvested from leukemic C3H(f) mice. Separation of bound from free labeled virus was effected using the double antibody method. The assay can detect fewer than 10(8) virus particles and has been used to measure the viral content of individual organs from inoculated leukemic C3H(f) mice and from Ak mice with spontaneous leukemia. Organs from noninoculated healthy C3H(f) mice crossreacted poorly in the system, virus generally being detectable only in the thymus and spleen and at low concentration. In some of the inoculated C3H(f) leukemic mice the viral content of as little as 0.5 mul of plasma is measurable. That this assay is for intact virus and not for soluble antigens of the viral envelope was proven by the observation that the immunoreactive material of plasma and extracts from thymus and liver of leukemic mice has a buoyant denisty in sucrose of 1.17-1.18 g/ml, corresponding to that of intact virus grown in tissue culture. With this sensitivity it may now be possible to quantitate viral concentrations in tissue and body fluids from the time of inoculation through the development of obvious pathology.
Experimental myasthenia in Balb/c mice immunized with rat acetylcholine receptor from rat denervated muscle.
Authors: Granato D A , Fulpius B W , Moody J F
Published: May 2006

A new model of an autoimmune disease of the neuromuscular junction was obtained by injection of acetylcholine receptor purified from rat denervated muscles into Balb/c mice. Anti-rat, then anti-mouse acetylcholine receptor antibodies, appear in mouse serum during the immunization procedure. Electrophysiological investigations performed on immunized mice reveal a neuromuscular block similar to that found in myasthenia gravis. Not a single mouse with objective signs of muscular weakness was lacking anti-mouse acetylcholine receptor antibodies but no correlation was found between their level and the severity of the disease.
A possible conformation for double-stranded polynucleotides.
Authors: Rodley G A , Scobie R S , Bates R H , Lewitt R M
Published: May 2006

A model is presented for double-stranded polynucleotides which involves side-by-side meshing of the two strands rather than double helical intertwining. The sugar-phosphate backbone has a twisted strip-like character, yet base-pairing of the Watson-Crick type is still possible. Structural features of the basic model are described and coordinates are presented for a representative example. The structure has, on the whole, reasonable sterochemical contacts, and can be shown to produce a fiber diffraction pattern with x-rays not unlike that of the B form of DNA.
Lateral hapten mobility and immunochemistry of model membranes.
Authors: Brûlet P , McConnell H M
Published: May 2006

A study has been made of the interaction of specific anti-nitroxide (anti-spin label) IgG antibodies, Fab fragments, complement, and liposomes containing dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and dipalmitoylphosphatidylcholine plus 0-50 mole % cholesterol, together with various concentrations of a head group spin-labeled phospholipid (0.5-0.01 mole % in the plane of the membrane). The spin-labeled lipid is the amide formed from the reaction of an iodoacetamide spin label, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide, with dipalmitoylphosphatidylethanolamine. We have reached two conclusions: (i) complement fixation depends on the lateral mobility of the lipid hapten at low hapten concentrations in the plane of the membrane (less than or equal 0.1 mole %) and does not depend strongly on this mobility at high hapten concentrations; (ii) cholesterol may have two distinct effects on complement fixation, one arising from an enhancement of hapten exposure to antibody binding sites and a second due to a modulation of membrane fluidity.
Fate of mismatched base-pair regions in polyoma heteroduplex DNA during infection of mouse cells.
Authors: Miller L K , Cooke B E , Fried M
Published: May 2006

Heteroduplex DNA has been constructed from two variants of polyoma virus that differ genotypically at four distinct sites. The genotypes of the progeny virus derived from infections of mouse cells with single heteroduplexes have been analyzed to determine how the genotypic markers of the parental heteroduplex segregate. Markers that are separated by a length of DNA greater than 600 nucleotides segregate independently. Segregation was not detected between two markers separated by only approximately 90 nucleotides. We interpret these results on the basis of the correction of mismatched base-pair regions in the heteroduplex before the completion of DNA replication. We suggest that this technique provides valuable information concerning gene conversion in mamalian cells and permits the transfer of genotypic markers from one virus strain to another.
Transient accumulation of Okazaki fragments as a result of uracil incorporation into nascent DNA.
Authors: Tye B K , Nyman P O , Lehman I R , Hochhauser S , Weiss B
Published: May 2006

Strains of Escherichia coli with a mutation in the sof (dnaS) locus show a higher than normal frequency of recombination (are hyper rec) and incorporate label into short (4-5S) DNA fragments following brief [3H]thymidine pulses [Konrad and Lehman, Proc. Natl. Acad. Sci. USA 72, 2150 (1975)]. These mutant strains have now been found to be defective in deoxyuridinetriphosphate diphosphohydrolase (dUTPase; deoxyuridinetriphosphatase, EC 3.6.1.23), the enzyme that catalyzes the hydrolysis of dUTP to dUMP and PPi. Reversion of one sof- mutation to sof+ restores dUTPase activity and abolishes the accumulation of labeled 4-5S DNA fragments. Mutants initially isolated as defective in dUTPase (dut-) are also hyper rec and show transient accumulation of short DNA fragments. Both the sof and dut mutations are located at 81 min on the E. coli map, closely linked to the pyrE locus. The sof and dut loci thus appear to be identical. A decrease in dUTPase as a consequence of a sof or dut mutation may result in the increased incorporation of uracil into DNA. Rapid removal of the uracil by an excision-repair process could then lead to the transient accumulation of short DNA fragments. It is possible that at least a portion of the Okazaki fragments seen in wild-type cells may originate in this way.
Density of sodium channels in mammalian myelinated nerve fibers and nature of the axonal membrane under the myelin sheath.
Authors: Ritchie J M , Rogart R B
Published: May 2006

The density of sodium channels in mammalian myelinated fibers has been estimated from measurements of the binding of [3H]saxitoxin to rabbit sciatic nerve. Binding both to intact and to homogenized nerve consists of a linear, nonspecific, component and a saturable component that represents binding to the sodium channel. The maximum saturable binding capacity in intact nerve is 19.9 +/- 1.9 fmol-mg wet-1; the equilibrium dissociation constant, Kt, is 3.4 +/- 2.0 nM. Homogenization makes little difference, the maximum binding capacity being 19.9 +/- 1.5 fmol-mg wet-1 with Kt = 1.3 +/- 0.7 nM. These values correspond to a density of about 700,000 sodium channels per node--i.e., about 12,000 per mum2 of nodal membrane. From the difference between the values of maximum saturable binding capacity in intact and homogenized preparation, given the statistical uncertainty of their estimate, it seems that the internodal membrane can have no more than about 25 channels per mum2. The significance of these findings for saltatory conduction and in demyelinating disease is discussed.
Nuclear coupling of 33S and the nature of free radicals in irradiated crystals of N-acetyl-L-cysteine.
Authors: Hadley J H , Gordy W
Published: May 2006

Hyperfine structure due to 33S in its natural abundance of 0.76% has been measured in the electron spin resonance of free radicals produced by x-irradiation of single crystals of N-acetyl-L-cysteine at 77 K. These measurements proved that the radicals produced at 77 K with principal g values of 1.990, 2.006, and 2.214 are monosulfide radicals with the 3p unpaired electron density of 0.70 on the S. They are believed to be negatively charged molecules RCH2S-H or neutral RCH2SH2 radicals in which 90% of the spin density of the captured electron is concentrated in a d-p hybrid orbital on the S. As the temperature is raised to 300 K, these, as well as the carbon-centered radicals produced at the lower temperature, are mostly converted to neutral disulfide radicals RCH2SS like those observed in irradiated cystine.
Cell differentiation in Dictyostelium under submerged conditions.
Authors: Sternfeld J , Bonner J T
Published: May 2006

Hitherto it has not been possible to obtain spore and stalk cell differentiation of the cellular slime molds in submerged cultures. It is shown here that cells, when placed in roller tubes under an atmosphere of oxygen, will form clumps and differentiate in 48-72 hr into mature spores and stalk cells. Although differentiation occurs without the normal morphogenetic movements, there is the appearance of an anteroposterior polarity of the cells in the clump. In addition to oxygen we examined a number of other factors that affect differentiation.
Structure and properties of a hybrid tryptophan synthetase of alpha chain produced by genetic exchange between Escherichia coli and Salmonella typhimurium.
Authors: Yanofsky C , Li S S , Horn V , Rowe J
Published: May 2006

Genetic exchange between the structural genes for the alpha chain of tryptophan synthetase [tryptophan synthase; L-serine hydro-lyase (adding indoleglycerol-phosphate), EC 4.2.1.20] of E. coli and S. typhimurium yielded recombinant genes that specified functional hybrid polypeptides. The alpha chains produced by three recombinants appeared to be identical but differed from those of E. coli and S. typhimurium by at least 27 and 8 amino acid residues, respectively. In vivo and in vitro tests of enzyme function suggest that the hybrid alpha chains are near-equivalent to their fully active parental proteins.
Nonspecific DNA binding of genome-regulating proteins as a biological control mechanism: Measurement of DNA-bound
Escherichia coli lac
repressor
in vivo
Authors: Kao-Huang Ying , Revzin Arnold , Butler Andrew P. , O'Conner Pamela , Noble Daniel W. , Von Hippel Peter H.
Published: May 2006

Binding of genome regulatory proteins to nonspecific DNA sites may play an important role in controlling the thermodynamics and kinetics of the interactions of these proteins with their specific target DNA sequences. An estimate of the fraction of
Escherichia coli lac
repressor molecules bound
in vivo
to the operator region and to nonoperator sites on the
E. coli
chromosome is derived by measurement of the distribution of repressor between a minicell-producing
E. coli
strain (P678-54) and the DNA-free minicells derived therefrom. Assuming the minicell cytoplasm to be representative of that of the parent
E. coli
cells, we find that less than 10% of the repressor tetramers of the average cell are free in solution; the remainder are presumed to be bound to the bacterial chromosome. The minimum
in vivo
value of the association constant for repressor to bulk nonoperator DNA (
K
RD
) calculated from these results is about 10
3
M
-1
, and analysis of the sources of error in the minicell experiment suggests that the actual
in vivo
value of
K
RD
could be substantially greater. The value of
K
RD
, coupled with
in vitro
data on the ionic strength dependence of this parameter, can be used to estimate that the
effective
intracellular cation activity of
E. coli
is no greater than about 0.24 M (and probably no less than 0.17 M) in terms of sodium ion equivalents. The minicell distribution experiments also confirm that the association constant for the binding of inducer-repressor complex to bulk nonoperator DNA (
K
RID
) is [unk]
K
RD
in vivo
. These results are used to calculate
minimum in vivo
values of
K
RO
and
K
RIO
(association constants for repressor and for inducer-repressor complex binding to operator) of about 10
12
M
-1
and about 10
9
M
-1
, respectively. The results fit a quantitative model for operon regulation in which nonspecific DNA-repressor complexes play a key role in determining basal and constitutive levels of gene expression [von Hippel, P. H., Revzin, A., Gross, C. A. & Wang, A. C. (1974)
Proc. Natl. Acad. Sci. USA
71, 4808-4812].
Developmental defects associated with glucosamine auxotrophy in
Saccharomyces cerevisiae
Authors: Ballou Clinton E. , Maitra Shyamal K. , Walker Jeffery W. , Whelan William L.
Published: May 2006

Saccharomyces cerevisiae
mutants, unable to make D-glucosamine owing to a defect in the enzyme 2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (amino-transferring) (EC 5.3.1.19), show aberrations both in sporulation and in vegetative growth. They grow normally on a medium of yeast extract, peptone, and dextrose (YEPD) containing D-glucosamine (1 mg/ml), and such cells accumulate 4 to 5 times the amount of D-glucosamine present in wild-type cells cultured on YEPD alone. When such mutant cells are shifted to YEPD alone, they continue to increase in cell mass for about 10 hr (three to four cell cycles) and produce strings of beads in which the cells fail to separate. Although each of the “cells” contains a nucleus, electron micrographs of thin sections reveal that septation is defective apparently owing to the inability to synthesize chitin, which forms the primary septum in
S. cerevisiae
. The viability of such cultures drops rapidly after 3-5 hr, a fact attributable to lysis of the cells through wall defects in the septum region where gross disorganization is apparent. When the mutant cells grown on YEPD plus D-glucosamine are transferred to sporulation medium (1% potassium acetate), they proceed through meiosis to produce viable spores that appear to be altered only in the nature of the spore wall. The spores lack a dark-staining surface layer that is visible in thin sections prepared from wild-type cells, they are notably less hydrophobic than wild-type spores, and they are digested and lysed by glucanases that do not affect normal spores. All of these properties suggest that D-glucosamine is required for spore maturation and is used to synthesize a glucanase-resistant hydrophobic surface layer on the primary glucan spore wall. In agreement with this postulate, D-glucosamine synthesis and the activity of the isomerase do not appear until late in meiosis when tetranucleate cells are abundantly present in the sporulation culture.
Cyclic AMP, the microtubule-microfilament system, and cancer.
Authors: Puck T T
Published: May 2006

Additional evidence is presented for the previously proposed existence in normal fibroblasts of a cyclic AMP-dependent network of microtubules and microfilaments, which is connected with cell membrane elements on one end and with nuclear structures on the other and whose disorganization leads to malignant transformation. In the presence of cyclic AMP derivatives sufficient to promote integrity of this network, cell growth limitation in suspension, increased transport of alpha-[14C]aminobutyrate, and the relatively tranquilized membrane of the normal fibroblast are also achieved. A pattern of distribution of actin and tubulin has been demonstrated showing aggregated actin deposits which are presumably responsible for the oscillatory knob activity of cells with the transformed habitus. Specific orientations of microtubular and filamentous elements with respect to the nucleus can be demonstrated. The hypothesis that the microtubular-microfilamentous structure conveys growth-regulatory information from the cell membrane to the nucleus and that its disorganization can lead to malignancy has been extended to explain various cellular manifestations.
Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange.
Authors: McMaster G K , Carmichael G G
Published: May 2006

We have developed a simple and rapid system for the denaturation of nucleic acids and their subsequent analysis by gel electrophoresis. RNA and DNA are denatured in 1 M glyoxal (ethanedial) and 50% (vol/vol) dimethyl sulfoxide, at 50 degrees. The glyoxalated nucleic acids are then subjected to electrophoresis through either acrylamide or agarose gels in a 10 mM sodium phosphate buffer at pH 7.0. When glyoxalated DNA molecules of known molecular weights are used as standards, accurate molecular weights for RNA are obtained. Furthermore, we have employed the metachromatic stain acridine orange for visualization of nucleic acids in gels. This dye interacts differently with double- and single-stranded polynucleotides, fluorescing green and red, respectively. By using these techniques, native and denatured DNA and RNA molecules can be analyzed on the same slab gel.
Proteolytic enzyme activation of rat ovarian adenylate cyclase
Authors: Richert Nancy D. , Ryan Robert J.
Published: May 2006

Various serine proteases (e.g., trypsin, α-chymotrypsin, Pronase, and subtilisin) stimulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in a membrane-enriched fraction of the rat ovary. Maximum stimulation is observed at protease concentrations ranging from 3 to 10 μg/ml. Higher protease concentrations inhibit ovarian adenylate cyclase in a dose-dependent manner. Protease stimulation causes a 6- to 8-fold increase in adenylate cyclase activity, which is comparable to the stimulation observed with human chorionic gonadotropin. Combinations of trypsin plus hormone or trypsin plus NaF stimulate ovarian adenylate cyclase activity to a greater extent than does any one of these alone.

The mechanism of protease stimulation of adenylate cyclase involves limited proteolysis because zymogen precursors fail to activate the cyclase as does trypsin pretreated with trypsin inhibitors. Unlike cholera toxin, the serine protease stimulation is immediate (within the first 5 min) and requires no additional factors (e.g., NAD
+
). It is unlikely that protease stimulation of adenylate cyclase results from a proteolytic modification of the hormone receptor on the cell surface, because of the additive effects noted above and because protease stimulation is also observed in ovaries desensitized to hormone that lack this hormone receptor. Results with Lubrol-treated membranes also suggest that proteolytic enzymes do not directly activate the catalytic subunit of the cyclase or unmask new catalytic sites because the protease effect (like hormonal stimulation) is abolished by the detergent, whereas fluoride stimulation is enhanced. Other data suggest that serine protease and chorionic gonadotropin stimulation of adenylate cyclase result from activation of a membrane protease that then regulates adenylate cyclase in the ovary.
Synapse turnover: the formation and termination of transient synapses.
Authors: Puro D G , De Mello F G , Nirenberg M
Published: May 2006

Neurons dissociated from chick embryo retina form synapses with cultured rat striated muscle cells in 35-90 min when neurite extension is uncoupled from later steps in synapse formation. The results suggest that a reaction is required for synapse formation after neurons adhere to muscle cells. All synapses between retina neurons and muscle cells are terminated in 3-10 days depending on the developmental age of the neurons. The half-lives of synapses between muscle cells and retina neurons from 8-, 12-, or 13-day embryos are 36, 26, and 5 hr and mean synapse life-times are 53, 37, and 7.1 hr, respectively. The results show that synapses turn over and that the rate of turnover increases during development. The results suggest that both synapse formation and termination rates are regulated and that the specificity of synaptic connections can be increased by selective termination of synapses.