bluuebunny/crossref_metadata_2025 huggingface dataset:
Estrogen induction of growth factors specific for hormone-responsive mammary, pituitary, and kidney tumor cells.
Authors: Sirbasku D A
Published: May 2006

The problem of estrogen-promoted tumor cell growth has been studied extensively in an attempt to establish the direct mitogenic role of these steroid hormones. We have developed cell lines from three estrogen-responsive tumors or cell populations: the H-301 kidney tumor cells established from a parent estrogen-dependent hamster kidney tumor, the GH3/C14 rat pituitary tumor cell line established as a subline of the original GH3 population, and the MTW9/PL mammary cell line developed from a parent estrogen- and prolactin-responsive MT-W9A carcinogen-induced rat tumor. With all three of these cell lines, we have encountered a paradox: although estrogens are obligatory for tumor formation in vivo, no direct mitogenic effect of estrogens can be shown in culture when assayed by an increase in cell number. We have thus considered the possibility that estrogens may induce growth factors in vivo that are then responsible for tumor formation by the three cell lines described. Experiments presented in this report show that extracts of rodent uterus, kidney, or liver contain growth activity for these three tumor cell lines, that estrogen treatment causes an increase in tissue content of these activities, and that the estrogen-induced activities are specific for the estrogen-responsive cells. These studies suggest that estrogen-responsive tumor growth in vivo includes the mechanism of estrogen leads to uterus, kidney, or liver leads to specific growth factors leads to estrogen-responsive tumor cells.
Proteins conferred by the virulence-specifying plasmid of Agrobacterium tumefaciens C-58.
Authors: Sonoki S , Kado C I
Published: May 2006

Membrane-associated and periplasmic proteins of Agrobacterium tumefaciens C-58 were compared with those from avirulent (nontumrigenic) derivative strains by slab and two-dimensional gel electrophoresis. Two proteins (Per-I and Per-2), with a molecular weight of 37,500 and 37,300, respectively, were detected in the supernatant fraction of cells of strain C-58 treated with EDTA and lysozyme in which a 117-megadalton plasmid confers virulence on the organism. The same proteins are missing in an avirulent plasmid-free derivative of C-58. When this derivative is mated with C-58, the resulting transconjugants regain the large C-58 plasmid together with the restoration of virulence and the expression of Per-1 and Per-2 proteins. When the transconjugants were cured of their plasmid, they concomitantly lost their virulence and Per-1 and Per-2. The functional roles of these proteins are unknown, but they are associated with the outer membrane and periplasmic fraction of the Agrobacterium cell. If directly involved in tumorigenesis, these proteins are not the sole determinants of tumorigenicity because they are synthesized in an avirulent derivative of C-58 that carries a deletion in the plasmid in the region conferring the tumorigenic phenotype. These results strongly suggest that the Per-1 and Per-2 proteins are plasmid-coded gene products. The possible roles of these proteins in specifying host range and host-cell attachment are also discussed.
AB variant of infantile GM2 gangliosidosis: deficiency of a factor necessary for stimulation of hexosaminidase A-catalyzed degradation of ganglioside GM2 and glycolipid GA2.
Authors: Conzelmann E , Sandhoff K
Published: May 2006

Human kidney extracts heated to 60 degrees and devoid of hexosaminidase activity (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase EC 3.2.1.30) stimulate more than 20-fold the hexosaminidase A-catalyzed degradation of ganglioside GM2 and of glycolipid GA2, the neuronal storage compounds of GM2 gangliosidosis. The stimulating factor of this extract, which is labile at temperatures above 60 degrees, is also present in kidney extracts from patients with infantile GM2 gangliosidosis having a deficiency of hexosaminidase A (Tay-Sachs disease, variant B) and a deficiency of hexosaminidases A and B (variant 0). Evidence is presented that this factor is defective in the AB-variant of infantile GM2 gangliosidosis which is characterized by an accumulation of glycolipids GM2 and GA2 despite the fact that the degrading enzymes, hexosaminidases A and B, retain normal activity levels. Thus, variant AB is an example of a fatal lipid storage disease that is caused not by a defect of a degrading enzyme but rather by a defective factor necessary for the interaction of lipid substrates and the water-soluble hydrolase.
Specific immunosuppression of experimental autoimmune myasthenia gravis by denatured acetylcholine receptor.
Authors: Bartfeld D , Fuchs S
Published: May 2006

Specific immunosuppression of experimental autoimmune myasthenia gravis (EAMG) was achieved by the use of a denatured preparation of the acetylcholine receptor (AcChoR) that did not in itself induce the disease. Torpedo californica AcChoR was irreversibly denatured by complete reduction and carboxymethylation in 6 M guanidine hydrochloride. Rabbits immunized with reduced carboxymethylated receptor (RCM-AcChoR) produced antibodies that reacted with both RCM-AcChoR and intact AcChoR. The specificity of anti-RCM-AcChoR antibodies is different from that of anti-AcChoR antibodies because the former are directed to only part of the antigenic determinants present in the intact receptor. RCM-AcChoR, which by itself is completely nonmyasthenic, was shown to be capable of both preventing the onset of EAMG and of reversing the clinical symptoms in myasthenic rabbits. In all cases the therapeutic effect of RCM-AcChoR administration on EAMG was accompanied by a change in the immunological specificity of the antibodies. The crossreactivity between AcChoR and RCM-AcChoR and the nonpathogenicity of RCM-AcChoR appear to be crucial in governing the specific immunosuppressive effects of RCM-AcChoR on EAMG.
Regulation of aldosterone secretion by the renin-angiotensin system during sodium restriction in rats.
Authors: Aguilera G , Catt K J
Published: May 2006

The role of angiotensin II as mediator of the aldosterone response to short periods of sodium restriction was studied in rats by administration of a converting enzyme inhibitor to block formation of the octapeptide throughout the duration of decreased sodium intake. In control animals, short-term sodium restriction caused increased levels of adrenal receptors for angiotensin II, with enhancement of early and late steps in aldosterone biosynthesis and elevation of plasma aldosterone concentration. Each of these changes induced by sodium deficiency was abolished during blockade of angiotensin II formation by continuous infusion of the converting enzyme inhibitor, SQ 14,225. The absolute dependence of adrenal glomerulosa cell responses on angiotensin II formation indicates that the renin-angiotensin system is the primary regulator of aldosterone secretion during physiological fluctuations in sodium intake.
Sterol requirement of Mycoplasma capricolum.
Authors: Odriozola J M , Waitzkin E , Smith T L , Bloch K
Published: May 2006

Mycoplasmas require an external source of sterol for growth. For Mycoplasma capricolum this requirement is met not only by cholesterol but also by the methylcholestane derivatives lanosterol, cycloartenol, 4,4-dimethylcholesterol, and 4beta-methylcholestanol. Cholesteryl methyl ether and 3alpha-methylcholestanol serve equally well as sterol supplements. None of the growth-supporting sterol derivatives tested was metabolically modified. The unusual acceptance of diverse cholestane derivatives by a mycoplasma species contrasts with the structural attributes thought to be necessary for sterol function in eukaryotic membranes.
Spectroscopic sensitivity of
linear
-benzoadenine nucleotides to divalent metal counterions, side chain conformations, micelles, and enzymes
Authors: VanDerLijn Pieter , Barrio Jorge R. , Leonard Nelson J.
Published: May 2006

From pK
a
data for
lin
-benzoadenosine 5′-mono-, 5′-di-, and 5′-triphosphates, which are fluorescent “stretched-out” analogues of adenine nucleotides, it was possible to designate the cases of interaction of phosphate with the heteroaromatic moiety. The addition of divalent metal cations or quaternary ammonium micelles diminishes the direct intramolecular interaction between the phosphate(s) and base and consequently brings the pK
a
values close to that of
lin
-benzoadenosine. Fluorescence spectroscopy was used to investigate the interaction of
lin
-benzoadenine nucleotides with Mg
2+
, Mn
2+
, and Co
2+
. The association constants for the formation of such complexes were obtained from measurements of steady-state fluorescence quenching. Phase and modulation measurements of the fluorescence lifetimes of
lin
-benzoadenine nucleotides as a function of Co
2+
concentration permitted determination of the static component of the quenching due to intramolecular complex formation. The association constants of the
lin
-benzoadenine nucleotides with all of the divalent metal ions studied were greater than those observed for the corresponding adenine nucleotides and were in the order:
lin
-benzo-ATP >
lin
-benzo-ADP >
lin
-benzo-AMP. Fourier transform
1
H NMR of
lin
-benzo-ATP in the presence of Co
2+
showed broadening of the aromatic proton signals, the 2-H signal (corresponding to the 8-H in ATP) being the most affected. Models are proposed to explain the phosphate-base interaction, the influence of metal ions on base protonation, and the intramo- the intramolecular quenching observed in the complexes due to paramagnetic ion (Co
2+
, Mn
2+
) and base interaction.
Transfer of duck cell DNA sequences to the nucleus of 3T3 cells by Rous sarcoma virus.
Authors: Baxt W G , Meinkoth J L
Published: May 2006

Peking duck cell nuclear DNA has no complementarity to RNA of Prague C Rous sarcoma virus (RSV). Upon infection of Peking duck cells by Prague C RSV, polynucleotide sequences complementary to Peking duck cell nuclear DNA can be detected in the high molecular weight RNA from progeny Peking duck cell passaged RSV (RSVPD) by RNA.DNA molecular hybridazation. When 3T3 cells are subsequently infected by RSVPD, polynucleotide sequences complementary to Peking duck cell nuclear DNA can be detected in the 3T3 cell nuclear DNA by RNA.DNA molecular hybridization. The potential consequences of the transfer of the Peking duck cell nuclear DNA from the avian to the murine cells are discussed.
DNA sequence from the histidine operon control region: seven histidine codons in a row.
Authors: Barnes W M
Published: May 2006

The DNA sequence of 250 base pairs preceding the first structural gene of the histidine operon of Salmonella typhimurium was determined by the dideoxy chain-termination method. Single-stranded DNA template was provided by an M13-histidine transducing phage constructed for the purpose by in vitro recombination. The termination site for the histidine leader RNA is identified by analogy with the trp operon leader termination sequence, and is 47 nucleotides before the start codon of the first structural gene G. Beginning 150 nucleotides before the end of the presumed leader RNA is a possible short protein-coding region with seven histidine codons in a row. It is proposed that the major mechanism of histodine operon control must involve a ribosome arrested at this run of histidine codons when histidine is limiting.
Spatiotemporal pattern formation in thin layers and membranes: critical focal size.
Authors: Nazarea A D
Published: May 2006

A theory for the determination of the focal nucleation size of oscillatory membrane (or thin layer) reactions is developed on the assumption that the focal domain is square planar. From this, one can estimate that, for a period of the oscillatory mode of, say, 5 min and lateral diffusion coefficient of approximately 10(-9) cm2/sec, the critical focal area would be of the order of 148 micrometer2, corresponding to a linear dimension of approximately 12.2 micrometer2. An alternative general expression for the critical focal size, involving explicitly the viscosity of the membrane, is also given.
Immobilized lipid in acetylcholine receptor-rich membranes from Torpedo marmorata.
Authors: Marsh D , Barrantes F J
Published: May 2006

The lipid environment of acetylcholine receptor-rich membranes from Torpedo marmorata has been studied with spin labels. The electron spin resonance spectra of both stearic acid and steroid probes in the membranes revealed an immobilized lipid component, in addition to the fluid component which is found in aqueous bilayer dispersions of the extracted lipids. The spin labels also cause a differential paramagnetic quenching of the intrinsic protein fluorescence of the membranes, which is sensitive to the action of cholinergic ligands and follows a modified Stern-Volmer law. Electron spin resonance difference spectroscopy shows that the protein-associated lipid is immobilized with respect to rotation both around and perpendicular to the long molecular axis, with correlation times : formula: (see text) approximately 50-70 ns. The proportion of lipid in the immobilized component is greater than calculated for a single boundary layer around the protein and corresponds more closely to the total interstitial lipid occupying the area between densely packed protein units in acetylcholine receptor-rich membranes.
Units of transcription for cytoplasmic RNA in mouse myeloma cells.
Authors: Giorno R , Sauerbier W
Published: May 2006

The size of transcription units for several of the abundant cytoplasmic mRNA species in mouse myeloma cells has been analyzed by the ultraviolet light mapping technique. Inactivation kinetics and target size analyses for production of the predominant RNA species indicate that the mRNAs originate in precursor molecules that are 2--14 times larger than the mature mRNA. This estimate of the size of the transcription unit may be a minimum one since it would not take into account promoter-distal sequences if these were not necessary for processing of the mRNA precursor.
Depression of cytidine 3':5'-cyclic monophosphate phosphodiesterase activity in developing tissues of guinea pigs.
Authors: Helfman D M , Brackett N L , Kuo J F
Published: May 2006

Changes in levels of the newly discovered cytidine 3':5'-cyclic monophosphate (cyclic CMP) phosphodiesterase in some representative tissues (cerebral cortex, kidney, intestine, liver, heart, and lung) of guinea pigs from various developmental stages (fetus, neonate, pup, and adult) were studied and compared with those of cyclic AMP and cyclic GMP phosphodiesterases in the same tissues. It was observed that the tissue levels of cyclic CMP phosphodiesterase were invariably the lowest in every one of the fetal tissues examined, the highest in the corresponding tissues from the pups and adult, with intermediate levels seen in some neonatal tissues. The patterns of the ontogenetic changes in levels of cyclic AMP and cyclic GMP phosphodiesterase activities, however, were variable and tissue specific. These findings suggest that the depressed cyclic CMP phosphodiesterase activity (hence, the elevated cyclic CMP concentration) is perhaps a common factor in developing tissues undergoing rapid cell proliferation. The data also suggest that metabolism of cyclic CMP is perhaps more closely related to cell proliferation than the metabolism of cyclic AMP or cyclic GMP.
Inhibition of growth of transformed cells and tumors by an endogenous acceptor of galactosyltransferase
Authors: Podolsky D. K. , Weiser M. M. , Isselbacher K. J.
Published: May 2006

A galactosyltransferase glycopeptide acceptor purified from human malignant effusions was tested for its effects on cell growth
in vitro
and
in vivo
. Addition of the glycopeptide to the media of cells growing in tissue culture caused a significant inhibition of attachment and growth of transformed cells but had minimal effect on nontransformed cells. Transformed hamster cells (BHKpy, BHKpygiv, NILpy) and human malignant cells (BT-20 human breast and pancreatic carcinoma cells) were killed by the addition of as little as 0.5 μg of acceptor (per ml of medium), while nontransformed counterparts did not show a significant change in growth or morphology.
In vivo
studies showed that the acceptor inhibited development and progression of tumors in hamsters inoculated with tumorigenic BHKpy cells. Growth of tumors was inhibited 69-94% in animals given 20 μg of acceptor subcutaneously and 39-67% when acceptor was given intraperitoneally at the time of tumor cell inoculation. Administration of the acceptor after the development of a palpable tumor (≈0.5 cm) caused a 60-85% reduction in growth rate and, in some cases, actual reduction in size and disappearance of palpable tumor. These studies demonstrate that a galactosyltransferase glycopeptide acceptor purified from human malignant effusions produces selective inhibition of transformed cell growth in animal and tissue culture systems.
H-2.28, an alloantigenic marker allelic to H-2.1, is expressed on all three known types of H-2 molecules.
Authors: Démant P , Iványi D , Neauport-Sautes C , Snoek M
Published: May 2006

Each allele at the K or D region of the H-2 complex produces two kinds of "allelic" or mutually exclusive antigenic characteristics: its unique private specificity and a public specificity(ies) of either the H-2.28 or H-2.1 family. The private specificities of the K and D regions are expressed on H-2K and H-2D molecules, respectively. The D region produces another molecule, H-2L, which lacks the H-2K and H-2D private specificity but exhibits the H-2.28 or H-2.1 specificity. We analyzed the expression of the H-2.28 determinants on H-2K, H-2D, and H-2L molecules. When an antiserum against H-2.28 is used to sensitize cells where it can react with only H-2K molecules or H-2D molecules, by subsequent elution antibodies against H-2.28 are recovered that can also react with H-2L molecules. Hence, determinants reactive with antibodies against H-2.28 are present on H-2L as well as on H-2K and H-2D molecules. The expression of the H-2.28/H-2.1 polymorphism on all three known types of H-2 molecules, without some obvious relation to the private specificities, suggests that the antigenic determinants of these two kinds of allelic systems (private in contrast to H-2.28/H-2.1) may be controlled by separate genes, even when they are expressed on the same molecule.
A virus similar to human hepatitis B virus associated with hepatitis and hepatoma in woodchucks.
Authors: Summers J , Smolec J M , Snyder R
Published: May 2006

Particles with properties similar to those associated with human hepatitis B were found in serum from woodchucks with chronic hepatitis and hepatocellular carcinoma. It is suggested that woodchuck hepatitis virus is a second member of a novel class of viruses represented by the human hepatitis B virus.
Mechanochemical properties of brain clathrin: interactions with actin and alpha-actinin and polymerization into basketlike structures or filaments.
Authors: Schook W , Puszkin S , Bloom W , Ores C , Kochwa S
Published: May 2006

Two molar urea (pH 7.5) and column chromatography on Sepharose 4B were used to separate clathrin (coat protein) from the membrane of coated vesicles from bovine brain. Lytron (polystyrene) particles were used for study of the interaction of clathrin with contractile proteins. Muscle G-actin, F-actin, and alpha-actinin were bound by clathrin-coated Lytron particles, while no interaction was found when muscle tropomyosin and serum albumin were tested. Clathrin molecules dispersed in a solution of 20 mM Tris-HCl (pH 7.5) were found to be elongated. When the pH was adjusted from 7.5 to 6.5, clathrin molecules associated into basketlike or cage structures similar in size and shape to those observed in enriched preparations of coated vesicles. Below pH 6.0, cages or baskets became amorphous aggregates. Raising the pH from 6.5 to 8.0, addition of 5-10 mM ATP or EDTA, or addition of 200 mM KCl resulted in the dissassembly of baskets and the formation of filamentous arrays of various widths. Because of clathrin's biochemical and biophysical properties, its interaction with contractile proteins, and its presence in the membrane of vesicles of various cell types, we classified clathrin in the group of mechanochemical proteins.
Assembly of the mitochondrial membrane system: partial sequence of a mitochondrial ATPase gene in Saccharomyces cerevisiae.
Authors: Macino G , Tzagoloff A
Published: May 2006

The nucleotide sequence of mitochondrial DNA of a cytoplasmic "petite" mutant of Saccharomyces cerevisiae is reported. The DNA has a repeat length of 1060 base pairs and contains a genetic marker (oli-1) for the ATPase proteolipid. The nucleotide sequence reveals the presence of part of the structural gene of the subunit-9 proteolipid of the ATPase complex and an extended A+T-rich region adjacent to the carboxyl-terminal end of the gene. The structural gene sequence agrees with the primary structure of the protein. These studies point out the feasibility of using the DNA of appropriately marked "petite" mutants to obtain the sequence of mitochondrial genes.
Transcription of cloned Xenopus 5S RNA genes by X. laevis RNA polymerase III in reconstituted systems.
Authors: Ng S Y , Parker C S , Roeder R G
Published: May 2006

When incubated with a soluble extract from large oocytes of Xenopus laevis, recombinant DNA plasmids containing either X. laevis oocyte 5S DNA or X. borealis oocyte 5S DNA direct the synthesis of discrete 5S RNAs, which by size and sequence analysis are similar or identical to the corresponding 5S RNAs synthesized in vivo. Synthesis of the 5S RNAs is mediated by a soluble endogenous RNA polymerase III (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6), which presumably recognizes specific initiation and termination sites in the 5S genes. Optimal conditions for accurate synthesis and the kinetics of the reactions have been determined. A soluble postchromatin supernatant fraction has also been isolated from immature oocytes. Although devoid of a functional endogenous RNA polymerase III, this extract contains a component(s) that effects the accurate transcription of 5S genes (in a plasmid) by a purified RNA polymerase III.
Interaction of Escherichia coli RNA polymerase with promoters of several coliphage and plasmid DNAs.
Authors: von Gabain A , Bujard H
Published: May 2006

The interaction of Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) with restriction fragments obtained from various E. coli related DNAs was studied in vitro. The DNAs investigated included several coliphage genomes (T5, lambda, T7, fd) and plasmid DNAs (pML 21, pSC101). By using the nitrocellulose filter binding of the enzyme-DNA complexes, fragment-specific relative rates of complex formation as well as complex stabilities were determined. Promoter-specific relative rates of polymerase binding were derived from fragment-specific rates by taking into account the number of major binding sites for RNA polymerase within several DNAs. Estimates of the stability of complexes formed between some major binding sites and the enzyme were obtained by studying the rate of complex decay. Both characteristics--rate of complex formation and rate of decay--varied widely and independently of each other. The promoters reacting most efficiently with E. coli RNA polymerase were found in the early region of coliphage T5 whereas some promoters in pML 21, or for example, the lambda promoter PI, belong to signals binding the enzyme most slowly. Based on the second-order rate constant determined for the interaction of E. coli RNA polymerase with promoters of phage fd, the fastest promoters characterized so far reacted with rates in the order of 10(8) M-1s-1. The hierarchy of promoters established here is of interest from the viewpoint that promoter strength correlates with the rate of polymerase binding. Among the promoters studied here this rate spans a range of 2 orders of magnitude.
Helical repeat of DNA in solution.
Authors: Wang J C
Published: May 2006

The helical repeat of DNA in solution has been measured directly by analyzing the gel electrophoretic patterns of pairs of covalently closed DNAs with length differences between 1 and 58 base pairs, out of a total length of about 4350 base pairs per DNA molecule. The method is based on the observation that for a covalently closed DNA of a fixed size of n base pairs (n of the order of several thousand), under appropriate conditions, two topological isomers (topoisomers) differing by 1 in their linking numbers are well resolved by gel electrophoresis. If the size of the DNA is increased to n + x base pairs, unless x is an integral multiple of the helical repeat h, the bands of the topoisomers with n + x base pairs per molecule are all shifted relative to the bands of the topoisomers with n base pairs per molecule. The magnitude of the shift is directly related to the nonintegral residual of x/n. Analysis of the set with x ranging from 1 to 58 gives the DNA helix repeat in solution as 10.4 base pairs per turn under physiological conditions, with an estimated probable error of +/- 0.1. This result strongly supports the double helix structure of DNA and rejects the side-by-side model of Rodley et al. [Rodley, G.A., Scobie, R.S., Bates, R.H. T & Lewitt, R.M. (1976) Proc. Natl. Acad. Sci. USA 73, 2959-2963]. The helical repeat of DNA measured in solution is significantly different from the value 10.0 base pairs per turn for the B form fiber structure.
Resolution of adenylate cyclase sensitive and insensitive to Ca2+ and calcium-dependent regulatory protein (CDR) by CDR-sepharose affinity chromatography.
Authors: Westcott K R , La Porte D C , Storm D R
Published: May 2006

Partially purified adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from bovine brain cortex was fractionated into two separate forms by calcium-dependent regulatory protein (CDR)-Sepharose affinity chromatography. The major form of the enzyme, comprising approximately 80% of the applied activity, did not bind to the affinity column in the presence of Ca2+ and was insensitive to the CDR. Approximately 20% of adenylate cyclase activity was absorbed to CDR-Sepharose in the presence of Ca2+. This activity was stimulated by Ca2+ and CDR. This study directly demonstrates that brain cortex contains Ca2+-CDR-sensitive and -insensitive forms of adenylate cyclase and indicates that CDR-Sepharose may be a useful tool for purification of adenylate cyclase. The Ca2+ -stimulated adenylate cyclase was purified at least 55-fold with a 13% yield.
Biosynthesis and self-assembly of protein S, a development-specific protein of
Myxococcus xanthus
Authors: Inouye Masayori , Inouye Sumiko , Zusman David R.
Published: May 2006

Myxococcus xanthus
is a Gram-negative bacterium that has a complex life cycle including a temporal sequence of cellular aggregation, mound formation, and myxosporulation. During development, protein S (molecuar weight 23,000) is induced and accumulates in very large amounts. Protein S was found in the soluble fraction of early developmental extracts and in the insoluble fraction in later extracts. This insoluble form of protein S can be solubilized by the addition of 1 M NaCl at 0°C to extracts from aggregated cells (mound stage) or by the addition of 1 M NaCl at 30°C to mature spores. Salt extraction (1 M NaCl) of protein S from mature spores was partially inhibited by the addition of Mg
2+
and almost completely inhibited by the addition of Ca
2+
. The viability of spores was not changed by a salt extraction that removed their protein S. Examination of thin sections of mature spores and extracted spores by electron microscopy suggested that the protein S-deficient spores lacked a spore surface coat about 300 A thick. Purified protein S will spontaneously self-assemble onto protein S-deficient spores after removal of the NaCl by dialysis or by addition of 10 mM Ca
2+
to undialyzed samples. Glycerol-induced spores did not contain protein S and did not serve as primers for assembly of protein S. Quantitation of the self-assembly process showed almost stoichiometric binding of protein S to the protein S-deficient spores until saturation at 3.3 × 10
6
molecules per spore, a value 1.35 times higher than the normal level of proteins S found in mature spores. Protein S in the “reconstituted” spores was as protease resistant and sonication resistant as the protein S of native spores. Electron microscopy of the reconstituted spores revealed the assembly of new material on the spore surface. Adjacent spores were sometimes observed to be fused to each other through a common protein S layer. These results suggest that protein S serves a function in spore—spore interaction in the fruiting body.
Reconstitution of membranes and embryonic development in dissociated blastula cells of the sea urchin by reinsertion of aggregation-promoting membrane proteins extracted with butanol.
Authors: Noll H , Matranga V , Cascino D , Vittorelli L
Published: May 2006

Blastula embryos of the sea urchin Paracentrotus lividus, when dissociated into single cells by exposure to Ca2+- and Mg2+-free sea water, reassociate spontaneously to form aggregates capable of development to the final larval form (pluteus). This aggregation is prevented by Fab fragments obtained by immunization with purified membranes from blastula embryos. The inhibition was reversed by soluble proteins extracted with butanol from purified membranes or from intact cells. These extracts also strongly stimulated the rate of reaggregation of dissociated cells in the absence of Fab fragments. Exposure of dissociated cells to 2.5% (vol/vol) butanol removed completely the protein(s) responsible for reaggregation of the cells without impairing their viability. Reaggregation and embryonic development were completely restored to the extracted cells by readdition of the proteins extracted from either membranes or cells. Extracted cells from Paracentrotus could be reconstituted with proteins from Arbacia.
mRNA-directed biosynthesis of α subunit of thyrotropin: Translation in cell-free and whole-cell systems
Authors: Kourides Ione A. , Weintraub Bruce D.
Published: May 2006

mRNA from mouse thyrotropic pituitary tumors was translated in frog oocytes (a whole-cell system) and in wheat germ extract and reticulocyte lysate (cell-free systems) in the presence of [
35
S]methionine. Synthesized peptides related to thyrotropin were identified in the three systems by immunoprecipitation with subunit-specific antisera developed against the α subunit of ovine lutropin (luteinizing hormone) and the β subunit of bovine thyrotropin. In wheat germ extract and reticulocyte lysate, a single immunoprecipitable form of the α subunit of thyrotropin was synthesized with an apparent molecular weight of 14,000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. In the frog oocyte, three forms of immunoprecipitable α subunit of thyrotropin were synthesized with apparent molecular weights of 20,000, 14,000, and 10,000. The 20,000 form is similar to unlabeled rat pituitary standard α subunit and
35
S-labeled mouse tumor α subunit in cell cultures (20,000-21,000); thus, it may represent a precursor-cleaved and glycosylated form. The 14,000 form synthesized in all three systems probably represents the pre-α subunit of thyrotropin; the 10,000 form, synthesized only in the frog oocyte, could be a proteolytically cleaved but unglycosylated form. Because only the α subunit of thyrotropin was identified and no larger molecular weight immunoprecipitable form of either subunit was detected in any of the translation systems, α and β subunits of thyrotropin appear to be translated from separate mRNAs.
Involvement of spectrin in membrane fusion: induction of fusion in human erythrocyte ghosts by proteolytic enzymes and its inhibition by antispectrin antibody.
Authors: Lalazar A , Loyter A
Published: May 2006

In contrast to intact human erythrocytes, human erythrocyte ghosts can be agglutinated but not fused by Sendai virus. Membrane fusion can, however, be induced in virus-agglutinated erythrocyte ghosts by addition of proteolytic enzymes such as trypsin, papain, or Pronase. When erythrocyte ghosts were reacted with antispectrin antiserum, the antiserum inhibited both the induction of fusion and the proteolysis of the membrane spectrin. The correlation between the membrane fusion process and the membrane cytoskeleton is discussed.
Is the product of the src gene a promoter?
Authors: Bissell M J , Hatié C , Calvin M
Published: May 2006

Addition of a potent promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), to primary avian tendon or chicken embryo fibroblast cells infected with a temperature-sensitive mutant of Rous sarcoma virus produced a complete transformed phenocopy at the nonpermissive temperature by the criteria tested. While normal, uninfected cultures also shifted towards a transformed phenotype after TPA addition, they did not achieve the same degree of morphological and biochemical alteration seen in virus-infected, TPA-treated cells. It is proposed that viral carcinogenesis, despite its rapidity, may occur in two stages: an "initiation" step caused by expression of a part of viral genome other than src (or by integration) and a promotion step (itself a multistep process) caused by the activation of the src gene. The src gene product could be enhanced or replaced by other promoting agents.
Efficiency of truncation selection
Authors: Crow James F. , Kimura Motoo
Published: May 2006

Truncation selection is known to be the most efficient form of directional selection. When this is modified so that the fitness increases linearly over a range of one or two standard deviations of the value of the selected character, the efficiency is reduced, but not greatly. When truncation selection is compared to a system in which fitness is strictly proportional to the character value, the relative efficiency of truncation selection is given by
f(c)/σ
, in which
f(c)
is the ordinate of the frequency distribution at the truncation point and σ is the standard deviation of the character. It is shown, for mutations affecting viability in
Drosophila
, that truncation selection or reasonable departures therefrom can reduce the mutation load greatly. This may be one way to reconcile the very high mutation rate of such genes with a small mutation load. The truncation model with directional selection is appropriate for this situation because of the approximate additivity of these mutations. On the other hand, it is doubtful that this simple model can be applied to all genes affecting fitness, for which there are intermediate optima and antagonistic selection among components with negative correlations. Whether nature ranks and truncates, or approximates this behavior, is an empirical question, yet to be answered.
Genetic control of cobalamin binding in normal and mutant cells: Assignment of the gene for 5-methyltetrahydrofolate:L-homocysteine
S
-methyltransferase to human chromosome 1
Authors: Mellman Ira S. , Lin Pin-Fang , Ruddle Frank H. , Rosenberg Leon E.
Published: May 2006

When extracts prepared from cultured human or rodent fibroblasts grown in medium containing [
57
Co]cobalamin were analyzed by polyacrylamide gel electrophoresis, most of the intracellular radioactivity migrated with the activity of the cobalamin-dependent enzyme 5-methyltetrahydrofolate:L-homocysteine
S
-methyltransferase (EC 2.1.1.13). Because the rodent and human forms of this enzyme are electrophoretically different, we used the binding of [
57
Co]cobalamin to detect the presence of the human methyltransferase isozyme in rodent-human somatic cell hybrids. As expected, binding and methyltransferase activities were found to cosegregate, thus confirming genetically their electrophoretic identity. Accordingly, we examined the [
57
Co]cobalamin-binding patterns and human chromosome contents of a panel of 12 rodent-human hybrid clones, and concluded that the gene for the methyltransferase (designated
Mtr
) is located on human chromosome 1. Using this information, we probed the nature of the molecular defect exhibited by fibroblasts cultured from patients expressing the
cbl C
mutation. Although these cells are unable to associate newly taken up [
57
Co]cobalamin with the methyltransferase, hybrids of mouse L-cells and
cbl C
cells containing chromosome 1 show a “reappearance” of the human [
57
Co]cobalamin-methyltransferase. These results indicate that the
cbl C
mutation does not affect the methyltransferase apoprotein, but rather some metabolic step that must convert cobalamin to a chemical form capable of attaching to the enzyme.
Generation of cytolytic T lymphocytes after reovirus infection: role of S1 gene.
Authors: Finberg R , Weiner H L , Fields B N , Benacerraf B , Burakoff S J
Published: May 2006

Cytotoxic T lymphocytes (CTLs) can be generated if spleen cells from reovirus-infected mice are stimulated in vitro with syngeneic reovirus-infected cells. These cytolytic effector cells demonstrate: (i) serotype specificity (i.e., maximal cytolytic activity is observed on target cells infected with the serotype used to induce the CTLs) and (ii) H-2 restriction. The SI gene was shown to be the predominant viral gene determining the specificity of the cytotoxic T cells. This genome segment has previously been demonstrated to encode the viral hemagglutinin and determines reovirus cell tropism in the nervous system.
Concanavalin A stimulates phospholipid methylation and phosphatidylserine decarboxylation in rat mast cells.
Authors: Hirata F , Axelrod J , Crews F T
Published: May 2006

When histamine release from rat peritoneal mast cells is stimulated by concanavalin A, membrane phospholipids are methylated in the early stage of this process. Exogenously added phosphatidylserine enhances the concanavalin A-induced histamine release, and at the same time the lectin markedly stimulates the decarboxylation and methylation of phosphatidylserine. Within minutes after the addition of concanavalin A to rat mast cells, the newly methylated phospholipids begin to disappear and an increased formation of lysophosphatidylcholine is observed. When rat mast cells are treated with concanavalin A in the absence of Ca2+, phospholipid methylation is stimulated but no significant release of histamine is detected. The subsequent exposure of the pretreated cells to Ca2+ causes increased release of histamine and degradation of methylated phospholipids. The inhibition of either synthesis or degradation of methylated phospholipids results in the inhibition of histamine release. These observations suggest that the synthesis and degradation of methylated lipids are an intrinsic part of the biochemical mechanism that modulate histamine release from mast cells.
Assumed initiation site of fibroin gene transcription.
Authors: Tsuda M , Ohshima Y , Suzuki Y
Published: May 2006

To map the initiation site of fibroin gene transcription on cloned genomic fibroin DNA (pFb29), we looked for precursors of fibroin mRNA, first by applying the hybridization mapping method to the RNA from the posterior silk gland of Bombyx mori. The 6.2-kilobase (kb) DNA fragment used for the assay, from 5.7 kb upstream to 0.5 kb downstream from the cap site, was end-labeled at the Xho I site (position +514) within the intervening sequence of the fibroin gene. By analyzing the labeled DNA protected by the RNA from nuclease S1 treatment, we have shown that the intervening sequence is transcribed together with the surrounding gene sequences in the posterior silk gland (producer) but not in the middle silk gland (nonproducer). Large transcripts that could include entire intervening (1 kb) and fibroin mRNA (16 kb) sequences were found. The 5' end of such transcripts was located around or near the cap locus. Synthesis and analysis of the DNA complementary to the transcripts primed within the intervening sequence region also confirmed that the 5' end of the transcripts is near the cap locus. These results strengthen the possibility that the cap site is the transcription initiation site of fibroin gene.
Role of mammalian RNase inhibitor in cell-free protein synthesis
Authors: Scheele George , Blackburn Peter
Published: May 2006

Addition of the human placental RNase inhibitor at 10 μ/ml to a mixture of wheat germ extract and translation components, prior to the addition of mRNA from dog pancreas or influenza virus-infected cells, resulted in a significant increase in the yield of proteins synthesized. Analysis of the translation products by sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the inhibitor preferentially increased the yield of the larger proteins. In the presence of the inhibitor, yields of the preprocarboxypeptidases were increased 4.5-fold and yields of preamylase were increased 15-fold. Incubation of the wheat germ extract or individual translation components with dog pancreas mRNA, with or without the placental inhibitor, indicated significant RNase contamination among the fractions. Two other
in vitro
protein synthesis systems—the reticulocyte lysate system and the Krebs ascites system—were found to contain latent RNase activity (RNase in complex with the inhibitor) and an excess of RNase inhibitor. The addition of placental RNase inhibitor did not increase the yield in these systems, except in those cases in which the RNase contamination approached the amount of endogenous inhibitor. When used during the isolation of rat liver cell fractions, the placental inhibitor increased the yield (as measured by
A
260
) of rough microsomes and detached polysomes by 24% and 4.6-fold, respectively. Analysis of translation products indicated that detached polysomes isolated in the presence of the inhibitor were intact; those isolated in the absence of inhibitor were degraded.
Primary structure of major outer membrane protein I of Escherichia coli B/r.
Authors: Chen R , Krämer C , Schmidmayr W , Henning U
Published: May 2006

The amino acid sequence of the pore-forming outer membrane protein I (porin) from Escherichia coli B/r has been determined. The polypeptide contains 340 amino acid residues resulting in a molecular weight of 37,205. The transmembrane polypeptide has no stretches of nonpolar residues, uninterrupted by charged side chains, longer than 11 amino acid residues. Regarding polarity, the chain can be subdivided into three regions: a distinctly hydrophilic region between residues 1 and 82 (51.2% polarity), a fairly nonpolar region between residues 83 and 194 (33.9% polarity), and a more hydrophilic region up to the COOH terminus (48% polarity). These results are interpreted as evidence against a simple transmembrane structure in which the membrane is spanned by a single contiguous sequence of hydrophobic amino acids, as has been proposed, for example, for glycophorin.
Squalene synthetase activity in human fibroblasts: Regulation via the low density lipoprotein receptor
Authors: Faust Jerry R. , Goldstein Joseph L. , Brown Michael S.
Published: May 2006

Squalene synthetase (farnesyltransferase; farnesyl diphosphate:farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21), the enzyme in the cholesterol biosynthetic pathway that converts farnesyl pyrophosphate into squalene, is subject to regulation in cultured human fibroblasts. When cholesterol-carrying low density lipoprotein (LDL) was removed from the serum of the culture medium, squalene synthetase activity increased 8-fold over 24 hr. When LDL was added back to the medium, squalene synthetase was slowly suppressed, 50% and 90% reduction occurring in 15 and 48 hr, respectively. Suppression of squalene synthetase required uptake of LDL via the LDL receptor; hence, it did not occur in mutant fibroblasts from a patient with homozygous familial hypercholesterolemia that lack receptors. The addition of a mixture of 25-hydroxycholesterol and cholesterol suppressed squalene synthetase equally well in normal and mutant fibroblasts. Coupled with previous data, the current findings indicate that cholesterol derived from LDL regulates at least two enzymes in the cholesterol synthetic pathway in fibroblasts: (
i
) its primary action is to rapidly suppress 3-hydroxy-3-methylglutaryl coenzyme A reductase [mevalonate:NADP
+
, oxidoreductase (CoA-acylating), EC 1.1.1.34], which reduces mevalonate production by 95% within 8 hr, and (
ii
) its secondary action is to slowly suppress squalene synthetase. The LDL-mediated suppression of squalene synthetase does not regulate
de novo
cholesterol synthesis; it occurs after 3-hydroxy-3-methylglutaryl coenzyme A reductase is already suppressed. Rather, we hypothesize that it may function to allow the pool size of farnesyl pyrophosphate to be maintained in the presence of LDL so that low levels of mevalonate can be shunted preferentially into nonsterol products, such as ubiquinone-10 and dolichol. This mechanism may explain the earlier observation that the synthesis of ubiquinone-10 in fibroblasts proceeds at a normal rate in the presence of LDL despite a 95% decrease in mevalonate production.
RNA polymerase III transcriptional units are interspersed among human non-alpha-globin genes.
Authors: Duncan C , Biro P A , Choudary P V , Elder J T , Wang R R , Forget B G , de Riel J K , Weissman S M
Published: May 2006

Cloned human DNA fragments containing globin genes are transcribed in vitro to form discrete RNA species. One transcription unit is located approximately 1500 base pairs upstream from the G-gamma-globin gene. This transcript is partially homologous to a polymerase III template located approximately 1000 base pairs upstream from the delta-globin gene and to DNA located a short distance downstream from the beta-globin gene.
Triplet states in photosystem I of spinach chloroplasts and subchloroplast particles.
Authors: Frank H A , McLean M B , Sauer K
Published: May 2006

We report light-induced electron paramagnetic resonance triplet spectra from samples of chloroplasts or digitonin photosystem I particles that depend upon the dark redox state of the bound acceptors of photosystem I. If the reaction centers are prepared in the redox state P-700 A X- FdB-FdA-, then upon illumination at 11K we observe a polarized chlorophyll triplet species which we interpret as arising from radical pair recombination between P-700+ and A-. This chlorophyll triplet is apparently the analog of the PR state of photosynthetic bacteria [Parson, W.W. & Cogdell, R.J. (1975) Biochim. Biophys. Acta 416, 105-149]. If the reaction centers are prepared in the dark redox state P-700 A X FdB-FdA-, then upon illumination at 11K we observe a different triplet species of uncertain origin, possibly pheophytin or carotenoid. This species is closely associated with the photosystem I reaction center and it traps excitation when P-700 is oxidized.
Long-term culture of capillary endothelial cells.
Authors: Folkman J , Haudenschild C C , Zetter B R
Published: May 2006

Capillary endothelial cells from rats, calves, and humans, have been carried in long-term culture. Bovine capillary endothelial cells have been cloned and maintained by serial passage for longer than 8 months. This prolonged culture was accomplished by using tumor-conditioned medium, gelatin-coated plates, and a method of enriching cells in primary culture. Cultured bovine capillary endothelial cells produce Factor VIII antigen and angiotensin-converting enzyme, but do not have Weibel-Palade bodies. Human cells do contain Weibel-Palade bodies. Capillary endothelial cells are distinguished from aortic endothelial cells by their requirement for conditioned medium. Bovine capillary endothelial cells in regular medium grow slowly with a mean doubling time of 67 hr and eventually die. In tumor-conditioned medium, these cells grow rapidly with a doubling time of 28 hr and continue to proliferate for as long as the tumor-conditioned medium is present. In contrast, bovine aortic endothelial cells grow as rapidly in regular medium as in tumor-conditioned medium. This method allows the production of pure capillary endothelial cells that may prove useful for studies of tumor angiogenesis, metastatic mechanisms, and the role of capillary endothelium in other pathologic states.