bluuebunny/crossref_metadata_2025 huggingface dataset:
Net uptake of gamma-aminobutyric acid by a high-affinity system of rat brain synaptosomes.
Authors: Pastuszko A , Wilson D F , Erecińska M
Published: May 2006

Rat brain synaptosomes isolated on discontinuous Ficoll gradient carry out rapid net uptake of gamma-aminobutyric acid through a high-affinity system (Km = 6.25 microM; Vmax = 1.2 nmol/min per mg of protein). The uptake of the labeled neurotransmitter is dependent on sodium concentration and is abolished by addition of 40 microM veratridine or 0.5 mM 2,4-diaminobutyric acid. Homoexchange in this preparation accounts for less than 10% of the measured uptake of gamma-amino[14C]butyric acid. It is concluded that the high-affinity transport exhibits properties characteristic of a system that is responsible for the rapid removal of gamma-aminobutyric acid from the synaptic cleft after neuronal transmission.
Identification and kinetic properties of the current through a single Na+ channel.
Authors: Fukushima Y
Published: May 2006

The kinetic properties of a single Na+ channel in the tunicate egg cell membrane were studied by the patch-recording technique. A conventional micropipette filled with a solution of 600 mM NaCl and 1.5 mM MnCl2 was used as the patch electrode. The seal resistance between patch electrode and egg surface was more than 1000 M omega. In the patch recording, the current fluctuations at a given membrane potential consisted of pulse-like events of a uniform amplitude. The amplitude was 1 pA at -56 mV and decreased as the membrane potential was made more positive. It is suggested that the fluctuation in the patch current is the current through a single Na+ channel for the following reasons. First, the reversal potential of the pulse-like fluctuation in the patch current was approximately equal to that of the total membrane Na+ current in a solution equivalent to that in the patch electrode. Second, the charges transferred by the patch current and by the total membrane Na+ current during a 170-msec command pulse showed parallel dependence on membrane potential. Third, the kinetic properties of the pulse-like fluctuations in the patch current were analyzed according to the Hodgkin-Huxley model, and it was shown that the time sequences of the pulse-like events were compatible with those for a single Na+ channel.
Requirement for nonoligodendrocyte cell signals for enhanced myelinogenic gene expression in long-term cultures of purified rat oligodendrocytes.
Authors: Bhat S , Barbarese E , Pfeiffer S E
Published: May 2006

Comparisons have been made of myelinogenic activities in fetal rat brain mixed primary cultures and cultures of isolated oligodendrocytes of comparable age. The specific activities of the sulfatide synthesis, 2',3'-cyclic-nucleotide 3'-phosphohydrolase (2',3'-cyclic-nucleotide 3'-phosphodiesterase, EC 3.1.4.37), and accumulation of myelin basic protein, when expressed per mg of protein, were as high (or generally higher) in isolated oligodendrocyte cultures as in comparable mixed primary cultures at 29 days. However, when these data were analyzed per oligodendrocyte, it became apparent that the isolated oligodendrocytes were substantially less active than their mixed culture counterparts. The results suggest the necessity of nonologodendrocyte positive signals for the optimal expression by oligodendrocytes of myelin-related differentiated functions. The isolation method involves the selection of oligodendrocytes by shaking them from primary cultures of rat brain, followed by the lysis of other contaminating cells in a balanced salt solution at pH 7.2. More than 99% of the isolated cells are viable, at least initially divide, and can be cultured for at least 60 days. The oligodendrocytes selected in this way were characterized by: (i) morphology, (ii) immunofluorescence labeling by antibodies to myelin basic protein and galactosylceramide, and (iii) biochemical analyses for myelin basic protein, activity of 2',3'-cyclic-nucleotide 3'-phosphohydrolase, and sulfogalactosylceramide synthesis.
Two calcium/calmodulin-dependent protein kinases, which are highly concentrated in brain, phosphorylate protein I at distinct sites.
Authors: Kennedy M B , Greengard P
Published: May 2006

Two calcium-stimulated protein kinase activities (ATP:protein phosphotransferase, EC 2.7.1.37) that phosphorylate protein I, a specific synaptic protein, have been identified in homogenates of rat brain. One of these is found in both the particulate and cytosolic fractions and phosphorylates a region of protein I that is phosphorylated in intact synaptosomes in response to calcium but not to cyclic AMP. The stimulation by calcium of the particulate enzyme and of the partially purified cytosolic enzyme requires the addition of calmodulin. It is not yet known whether the particulate and cytosolic enzymes are related. A second calcium-stimulated protein I kinase is found only in the cytosol and phosphorylates a region of protein I that is phosphorylated in intact synaptosomes in response to either calcium or cyclic AMP. The calcium stimulation of this latter kinase is probably mediated by calmodulin, judging from its inhibition by low concentrations of trifluoperazine. Both of the calcium-stimulated protein I kinases are more highly concentrated in brain than in other tissues. The two cytosolic kinases are distinguishable from each other and from myosin light chain kinase and phosphorylase b kinase by their substrate specificities and their chromatographic behavior on DEAE-cellulose.
Movement and site selection for priming by the primosome in phage phi X174 DNA replication.
Authors: Arai K , Low R L , Kornberg A
Published: May 2006

The Escherichia coli priming system used for initiation of DNA chains on phage phi X174 single-stranded DNA is a multiprotein unit called the primosome [Arai, K. & Kornberg. A. (1981) Proc. Natl. Acad. Sci. USA 78, 69-73]. Assembled with participation of seven prepriming proteins and primase at a unique place on the phi X174 DNA template, the primosome is bound tightly to the DNA, yet moves rapidly and unidirectionally opposite to primer and DNA chain synthesis. Contributions of protein n' and dnaB protein, two components of the primosome, to movement and site selection for priming are considered in this report. Figuratively, the primosome can be likened to a locomotive that depends on protein n' as its engine and dnaB protein as the engineer. Protein n', a DNA-dependent ATPase (dATPase) appears to use the energy of hydrolysis of the nucleoside triphosphate for processive translocation of the primosome. dnaB protein, A DNA-dependent ribonucleosidetriphosphatase, depends on allosteric effects of a nucleoside triphosphate to induce changes in the structure of the single-stranded DNA at preferred sequences that enable primase to synthesize a short primer for initiation of DNA synthesis (unpublished data). These primosome properties have important implications for the progress of the replication fork of the E. coli chromosome.
Specific transcription of mouse ribosomal DNA in a cell-free system that mimics control in vivo.
Authors: Grummt I
Published: May 2006

Cloned ribosomal DNA (rDNA) from mouse, which contains the initiation site of 45S pre-rRNA transcription and 5' flanking sequences, has been used as the template in an in vitro transcription system. In the presence of extracts from rapidly growing Ehrlich ascites cells, RNA polymerase I initiates specifically in that region of purified rDNA where the 5' end of 45S rRNA has been mapped. This is shown by electrophoretic analysis of the length of run-off transcripts synthesized from truncated templates, by S1 nuclease mapping, and by hybridization analysis of the in vitro products. The ability of the crude extracts to promote faithful transcription of mouse rDNA correlates with the proliferation rate of the cells. Only extracts prepared from exponentially growing mouse cells contain the factor(s) required for the faithful transcription of mouse ribosomal genes. Extracts from nongrowing or slowly growing mouse cells show very little activity. Thus, the cell-free system somehow reflects the rRNA synthetic activity of the cell and will prove valuable for the identification and purification of the various factors that are involved in the specific read-out of rDNA and may play a central role in the regulation of transcription of the ribosomal genes.
Quantitation of cation transport by reconstituted membrane vesicles containing purified acetylcholine receptor.
Authors: Wu W C , Moore H P , Raftery M A
Published: May 2006

A stopped-flow spectroscopic technique was used to study the kinetics of ion transport by reconstituted membrane preparations containing purified acetylcholine receptor. Influx of thallium (I) into membrane vesicles was monitored as a decrease, due to quenching by the thallous ion, in the fluorescence of an entrapped fluorophore. In a reproducible manner, the reconstituted receptor responded to cholinergic agonists by mediating rapid ion transport in the millisecond time range. The effect of agonists was blocked by receptor desensitization and by histrionicotoxin and was absent in membrane vesicles lacking receptor. Analysis of the fast kinetics of cation transport produced by saturating concentrations of agonists yielded an estimated rate of transport through a single reconstituted receptor channel. Comparison of this rate with those reported for in vivo preparations and for purified membranes shows that the reconstituted protein closely resembles the physiologically active receptor.
Kinetics and mechanism of heme-induced refolding of human α-globin
Authors: Leutzinger Yvonne , Beychok Sherman
Published: May 2006

Hemoglobin α and β chains are tightly packed, highly (75%) helical stable molecules. Removal of the heme results in unfolded (30% helical) unstable globin chains that can be refolded to the native conformation by recombination with heme. We have studied the kinetics of heme binding and the ensuing conformational changes by using three stopped-flow techniques: (
i
) fluorescence quenching, which monitors the spatial orientation and distance between the bound heme and the A12(14)α tryptophan; (
ii
) absorption at the Soret band maxima, whose position and intensity depend on the local environment of the heme and the nature of the axial ligands; and (
iii
) far-UV circular dichroism, which directly gauges the recovery of secondary structure. The fluorescence quenching was biphasic: An initial second-order decay, representing 80-85% of the total amplitude, marked the binding of hemin dicyanide to a relatively well-defined site at a rate constant of 3.3 × 10
7
M
-1
sec
-1
, corresponding to a half-time of 10 msec at 2.4 μM reactants. The Soret absorption and circular dichroism were also multiphasic, all three probes detecting a first-order process of half-time 25-40 sec, during which the final secondary and tertiary structures of the heme pocket were established, and the spatial relationship between the heme and the A12 tryptophan was fixed. A slower circular dichroism change, representing two-thirds of the total backbone refolding, with a half-time of 116 sec, marked the full acquisition of the native subunit conformation. The results show that the residues of the heme pocket achieve or closely approach their final three-dimensional structure well before the entire chain is folded. These measurements represent a direct observation of the rate of prosthetic group-induced secondary structure formation and illustrate the advantages of multiple probe analysis in outlining a protein folding pathway.
Identification of the glucagon receptor in rat liver membranes by photoaffinity crosslinking
Authors: Johnson Gary L. , MacAndrew Vincent I. , Pilch Paul F.
Published: May 2006

The photoaffinity crosslinker hydroxysuccinimidyl-
p
-azidobenzoate was used to attach
125
I-labeled glucagon covalently to a rat liver membrane protein of
M
r
≈ 53,000. Membranes that had been incubated with
125
I-labeled glucagon were treated in the dark with hydroxysuccinimidyl-
p
-azidobenzoate, and a covalent complex was then formed by irradiation with ultraviolet light. Characteristics of
125
I-labeled glucagon binding and covalent attachment to the
M
r
53,000 peptide were consistent with this peptide being a component of the glucagon receptor involved in the activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. Binding and covalent attachment of
125
I-labeled glucagon to the
M
r
53,000 peptide were inhibited by glucagon concentrations that were within the dose-response curve for adenylate cyclase activation, and GTP specifically decreased the photoaffinity crosslinking of
125
I-labeled glucagon to the
M
r
53,000 peptides. Insulin did not compete for the photoaffinity crosslinking of
125
I-labeled glucagon. The same technique of photoaffinity crosslinking that covalently attached
125
I-labeled glucagon to the
M
r
53,000 peptide with an efficiency of 1-2% can be used to attach
125
I-labeled insulin covalently to a
M
r
125,000 peptide with an efficiency of approximately 10%. This peptide has been shown to be a subunit of the high-affinity insulin-binding site in rat liver membranes. The technique of photoaffinity crosslinking with agents like hydroxysuccinimidyl-
p
-azidobenzoate provides a rapid, simple method of covalently attaching ligands to their putative receptors. Photoaffinity crosslinking does not require chemical modification of the labeled ligand and has a less stringent requirement for specific reactive groups than the commonly used bifunctional crosslinking reagents.
DNA sequence of the transfer RNA region of bacteriophage T4: implications for transfer RNA synthesis.
Authors: Mazzara G P , Plunkett G , McClain W H
Published: May 2006

Sequences encoding eight tRNAs and two stable RNAs of bacteriophage T4 are grouped together on the T4 genome in two clusters, separated by approximately 500 base pairs. The DNA sequence of part of this region was determined. Within each cluster coding sequences are separated by only one or a few base pairs. These findings imply that the RNAs may be processed from a single multimeric transcript, with initial endonucleolytic cleavages generating the previously characterized monomeric and dimeric precursors.
Myosin light chains and the developmental origin of fast muscle.
Authors: Stockdale F E , Raman N , Baden H
Published: May 2006

Physiological characteristics of embryonic and fetal fast muscle function are similar to those of adult slow muscles, whereas most biochemical data suggest that embryonic and fetal fast muscles contain only fast muscle myosin. In the studies reported here, myofibrillar preparations from developing avian pectoral muscle (fast muscle) were isolated and analyzed for myosin light-chain type and synthesis. These analyses show that early in development avian fast muscle synthesizes and assembles myofibrils with light chains of both slow and fast myosins. Later in development, fast muscle no longer assembles myofibrils containing slow myosin light chains due to the cessation of synthesis of slow myosin light chains in mid-development. These in vivo studies indicate that the more developmentally primitive type of skeletal muscle is one that synthesizes both slow and fast myosin light chains independent of its anatomic location, and an event(s) late in fast muscle development results in the repression of synthesis of slow myosin light chains.
15
N- and
2
H-substituted maleimide spin labels: Improved sensitivity and resolution for biological EPR studies
Authors: Beth A. H. , Venkataramu S. D. , Balasubramanian K. , Dalton L. R. , Robinson B. H. , Pearson D. E. , Park Charles R. , Park Jane H.
Published: May 2006

The resolution and sensitivity of electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) for biological applications are greatly improved by deuteration and substitution of
15
N for
14
N in the spin-labeled probe
N
-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (MSL). The EPR and ST-EPR spectra of the deuterated analogue [
2
H]MSL and the
15
N-substituted and deuterated derivative [
15
N,
2
H]MSL were compared with those of the parent MSL. The [
15
N,
2
H]MSL showed the greatest gain in sensitivity and the most marked sharpening of spectral features. These improvements were due to (
i
) a reduction in the spectral linewidths resulting from the relatively weak hyperfine interactions of the unpaired electron with deuterium and (
ii
) spectral simplification due to a reduction in the number of nuclear manifolds from three to two in replacing
14
N with
15
N. In the freely tumbling state, the spectra of [
15
N,
2
H]MSL and [
2
H]MSL showed 10-fold and 5-fold increases, respectively, in signal heights compared to MSL. To study the slow tumbling frequencies characteristic of biological molecules, the MSL and its derivatives were covalently bound to the enzyme glyceraldehyde-3-phosphate dehydrogenase [GAPDHaase; D-glyceraldehyde-3-phosphate:NAD
+
oxidoreductase (phosphorylating), EC 1.2.1.12] on cysteine-149 of the catalytic site. The EPR and ST-EPR spectra of [
15
N,
2
H]MSL and [
2
H]MSL adducts showed 3- and 1.5-fold gains in sensitivity, respectively. More important, there were striking increases in resolution, particularly for [
15
N,
2
H]MSL over MSL. These improvements were observed throughout the correlation time range from 0.1 μsec to 1 msec. The EPR spectrum of [
15
N,
2
H]MSL-GAPDHase at X-band showed no overlap of the two nuclear manifolds; therefore, all the elements of the A and
g
tensors could be measured directly from the spectrum. The increase in sensitivity and resolution of the
15
N- and deuterium-substituted spin labels permitted quantitative simulation of the EPR and ST-EPR spectra of a labeled protein. Computation time was reduced 90% by
15
N substitution. Use of
15
N-substituted and deuterated spin probes substantially improved characterization of the motional properties of a protein.
Stochastic flows in integral and fractal dimensions and morphogenesis
Authors: Hatlee Michael D. , Kozak John J.
Published: May 2006

The effect of dimensionality and spatial extent on the dynamics of an irreversible reaction confined to a finite system was studied by a Monte Carlo simulation. Stochastic flows on surfaces of integral and fractal dimensions and the consequences of reducing the dimensionality of the reaction space are described. As regards the timing and efficiency of chemical reactions in small systems, our simulations show that placing the reactive site at a central location may be favored at an early stage of growth but, as the system evolves in size, a location on the boundary becomes favored. The possible relevance of these calculations to the problem of morphogenesis is brought out.
Mathematical analysis of the chemosmotic polar diffusion of auxin through plant tissues
Authors: Goldsmith Mary Helen M. , Goldsmith Timothy H. , Martin Monroe H.
Published: May 2006

Equations have been developed to describe the diffusional movement of a weak acid such as the auxin indoleacetic acid through a long file of vacuolated cells, where cellular accumulation is driven by the pH gradients across the cell membranes. If the permeability to the auxin anion is greater at one end of the cell than at the other, diffusional movement takes the form of polar transport, which exhibits: a nearly constant velocity either for the front or for a pulse of radioactive auxin, the capacity to move auxin against an external gradient of concentration, and a polar ratio that increases exponentially with the length of the section. The determinants of velocity include both diffusion through the vacuole and permeation steps at the cell membranes. Except for the permeabilities of the membranes to the anion, values are now available for all of the physical parameters in the equations. With reasonable estimates of permeability coefficients for the anion, the equations predict a velocity of transport of about 1 cm hr
-1
, which agrees well with measured values. The analysis indicates, however, that the underlying cellular polarity may be greater than has been heretofore assumed. We thus demonstrate that the hypothesis of chemosmotic polar diffusion is capable of accounting quantitatively for the major features of auxin transport and provides a theoretical framework whose elements can be tested in future experiments.
Role of DNA gyrase subunits in synthesis of bacteriophage phi X174 viral DNA.
Authors: Hamatake R K , Mukai R , Hayashi M
Published: May 2006

The role of Escherichia coli DNA gyrase subunit A and subunit B during phi X174 viral DNA synthesis was investigated. Addition of nalidixic acid (an inhibitor of gyrase subunit A) and novobiocin (an inhibitor of gyrase subunit B) to an in vitro system capable of synthesizing phi X174 viral DNA inhibited DNA synthesis. The inhibition caused by novobiocin, however, was not due specifically to an inhibition of gyrase subunit B because DNA synthesis in an in vitro system composed of an extract containing novobiocin-resistant gyrase subunit B was also inhibited by novobiocin. The requirement for gyrase subunit A and the dispensability of gyrase subunit B during viral strand synthesis was confirmed in vivo by examining phi X174 viral DNA synthesis in host bacteria containing temperature-sensitive gyrase subunits.
Evidence that the Abelson virus protein functions in vivo as a protein kinase that phosphorylates tyrosine.
Authors: Sefton B M , Hunter T , Raschke W C
Published: May 2006

Both lymphocytes and fibroblasts that have been transformed by ABelson murine leukemia virus contain 6- to 12-fold increased levels of the rare modified amino acid phosphotyrosine in their proteins. This observation, coupled with the fact that the p120 protein encoded by this virus has been shown to undergo an apparent autophosphorylation to yield phosphotyrosine in vitro, suggests that Abelson virus encodes a protein kinase that phosphorylates tyrosine in transformed cells. These results are similar to those obtained previously with Rous sarcoma virus and suggest, by analogy, that the modification of cellular polypeptides through the phosphorylation of tyrosine may be involved in cellular transformation by Abelson virus. p120 isolated from transformed cells contains phosphoserine, phosphothreonine, and phosphotyrosine. The phosphotyrosine is found at two sites in the protein. p120 therefore may be a protein kinase that undergoes autophosphorylation in vivo.
Iodoazidobenzylpindolol, a photoaffinity probe for the beta-adrenergic receptor.
Authors: Rashidbaigi A , Ruoho A E
Published: May 2006

A high-affinity pindolol derivative, (+/-)-1-(indol-4-yloxy)-3-[1-(p-azido-m-iodophenyl)-2-isobutylamine]-2-propanol (IABP), has been prepared; it contains an iodide and an azide functional group and acts as a photoaffinity label for the beta-adrenergic receptor. When [125I]IABP (specific activity 1300 Ci/mmol) was photolyzed with crude duck erythrocyte membrane preparations, which contain beta-adrenergic receptor binding sites highly specific labeling of two polypeptides was observed upon electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. These two polypeptides, A (Mr approximately 45,000) and B (Mr approximately 48,500), and B (Mr approximately 48,500), were photolabeled in a ratio of approximately 4:1 A/B. Binding of [125I]IABP and covalent derivatization of the beta receptor was at least 70% specific. The data indicate that [125I]IABP is a very effective compound for identification of polypeptides containing the beta-receptor binding site even in crude membrane preparations. The beta receptor of the duck erythrocyte plasma membrane may be composed of more than one subunit.
Use of lectins and polyethylene glycol for fusion of glycolipid-containing liposomes with eukaryotic cells.
Authors: Szoka F , Magnusson K E , Wojcieszyn J , Hou Y , Derzko Z , Jacobson K
Published: May 2006

Efficient fusion of phospholipid vesicles with monolayer cultures of eukaryotic cells was accomplished by attaching glycolipid-containing vesicles to the cell surface by using a lectin displaying binding for both the cell surface and the glycolipid, followed by treatment with polyethylene glycol. Fusion was inferred from the transfer of fluorescent lipid analog probes embedded in the vesicle membrane over the entire cell surface and of fluoresceinated proteins from the aqueous space of the vesicle to the cytoplasm of the cell. Fluorescence recovery after photobleaching showed that both the injected membrane and the cytoplasmic markers were mobile. Two different lectin--glycolipid combinations [Ricinus communis agglutinin I-lactosylcerebroside and concanavalin A-tetradecyl- (or hexadecyl-) maltobionamide] were used to promote attachment of lipid vesicles before polyethylene glycol-induced fusion with BG-9 human fibroblasts, NIL-8M2 hamster cells, or L-929 mouse cells. In the absence of lectin or polyethylene glycol, fusion was negligible. However, when both the lectin and the glycol were used, a dramatic increase in the transfer of both vesicle membrane and aqueous space markers from the liposomes to the cells occurred.
Quantitative colony method for tumorigenic cells transformed by two distinct strains of Friend leukemia virus
Authors: Mager Dixie L. , Mak Tak W. , Bernstein Alan
Published: May 2006

An
in vitro
colony method capable of detecting spleen cells malignantly transformed by Friend leukemia virus is described. These colony-forming cells, which form large erythroid colonies (10
4
-10
5
cells) in methylcellulose, can be detected late after infection with either the anemia-inducing (FV-A) or polycythemia-inducing (FV-P) isolates of Friend virus. Colony formation by these cells is dependent only on fetal calf serum as an exogeneous growth factor. The presence of these colony-forming cells in FV-P-infected spleens could not be detected until at least 3 weeks after virus infection, even though the most rapid increase in spleen weight occurred earlier, between 1 and 2 weeks after infection. Thereafter, the numbers of colony-forming cells increased sharply up to 5 weeks after infection with FV-P, beyond which time the mice generally did not survive. After infection with FV-A, colony-forming cells were detected only at 8-12 weeks and their numbers generally increased thereafter. Permanent cell lines were established from a significant fraction of FV-P and FV-A-induced colonies, and these cell lines could be chemically induced to synthesize hemoglobin. All individual colonies produced complete Friend virus complex. However, virus production appeared to decline in at least some cell lines. Both FV-P- and FV-A-induced colonies contained cells capable of forming spleen colonies in irradiated recipients and subcutaneous tumors in unirradiated mice. Thus, the assay method described here appears to detect a unique class of malignant Friend virus-transformed cells that can be detected only in the advanced stages of Friend virus-induced erythroleukemia.
Increased nuclear conjugated polyamines and transglutaminase during liver regeneration.
Authors: Haddox M K , Russell D H
Published: May 2006

The nuclear content of conjugated polyamines increased during rat liver regeneration. Conjugated polyamines isolated from the acid-precipitable fraction of nuclei required peptide bond hydrolysis for release of the parent compounds. The most striking change occurred in conjugated putrescine which fluctuated in a biphasic manner; maximal nuclear levels 12-fold and 25-fold above those of sham-operated controls were achieved at 4 and 42 hr after hepatectomy, respectively. Conjugated spermidine and spermine increased 3- and 2-fold respectively within 4 hr and remained high throughout the 48 hr studied. When expressed on the basis of mg of nuclear protein, the maximal conjugated putrescine increased 19-fold, conjugated spermidine increased 2-fold, and conjugated spermine decreased by 50%. Therefore, the spermidine and spermine conjugates may be of a more constitutive nature whereas the large changes in the nuclear conjugation of putrescine associated with the onset of growth may play a regulatory role. The nucleus also contained transglutaminase (R-glutaminyl-peptide:amine gamma-glutamyl-yltransferase, EC 2.3.2.13), an enzyme shown in vitro to conjugate polyamines covalently to proteins. The specific activity of the nuclear enzyme increased rapidly after partial hepatectomy to a level 3-fold above control at 4 hr and 7-fold above control at 42 hr. The increased conjugating activity resulted from an increase in detectable maximal velocity and not a change in affinity of the enzyme for putrescine (Km congruent to 0.4 mM). There was also a 3-fold increase at 42 hr in the number of nuclear amine acceptor sites present to which radiolabeled putrescine could be conjugated by endogenous enzyme.
Regulatory interactions between mitochondrial genes: interactions between two mosaic genes.
Authors: Dhawale S , Hanson D K , Alexander N J , Perlman P S , Mahler H R
Published: May 2006

We have studied the mitochondrial DNA and the phenotypes of strains of Saccharomyces cerevisiae with specific intervening sequences in two mosaic genes: cob (the gene for apocytochrome b) and oxi3 (the gene for subunit I of cytochrome oxidase). The results suggest the following. (i) The presence of an intervening sequence downstream encompassing the intron box7 is sufficient for the regulation of oxi3 by cob (BOX phenotype); two sequences (containing intron loci box3 and box10) upstream in cob and two in oxi3 are dispensable. (ii) Strains without the two sequences upstream still contain the downstream sequence and the competence to specify a functional trans-acting element. Mutational lesions in this segment are phenotypically indistinguishable from box7 mutants, including the accumulation of polypeptides with homologous amino acid sequences. (iii) A catabolite-sensitive BOX phenotype, characteristic of mutants in the first exon, requires the simultaneous presence of an adjacent intervening sequence. A model is presented in which a hypothetical product specified by an intron (locus box7) of the cob gene controls the expression of a second mosaic gene (oxi3).
Reduced tumor growth after low-dose irradiation or immunization against blastic suppressor T cells.
Authors: Tilkin A F , Schaaf-Lafontaine N , Van Acker A , Boccadoro M , Urbain J
Published: May 2006

Suppressor T cells have been shown to be much more radiosensitive than other lymphoïd cells, and we have tried to reduce tumor growth by low-dose irradiation. Syngeneic DBA/2 mice received whole-body irradiation (150 rads; 1 rad = 0.01 J/kg) 6 days after P815 tumor inoculation. Tumor growth is significantly reduced in mildly irradiated mice. We also attempted to reduce syngeneic tumor growth by raising immunity against suppressor T cells in two different systems. DBA/2 mice were immunized against splenic T cells collected after disappearance of cytotoxicity and then injected with P815 tumor cells. These mice develop a very high primary cytotoxicity against P815 cells. C57BL/6 mice were immunized against blastic suppressor T cells, before injection of T2 tumor cells. Some of these mice reject the tumor and other develop smaller tumors than control mice. These results could be explained by the induction of antiidiotypic activity directed against the immunological receptors of suppressor T lymphocytes, because immunization with blastic suppressor T cells from mice bearing the T2 tumor does not modify the growth of another tumor, T10.
Identification of classical anaphylatoxin as the des-Arg form of the C5a molecule: Evidence of a modulator role for the oligosaccharide unit in human des-Arg
74
-C5a
Authors: Gerard Craig , Hugli Tony E.
Published: May 2006

A functionally active and potentially lethal fragment of the fifth component of complement (C5) is generated during complement activation in serum from animals of various species. This factor, termed the “classical” anaphylatoxin, was isolated from porcine serum and was identified chemically as the des-Arg derivative of the well-characterized C5a molecule. Unlike the C3a and C4a anaphylatoxins, porcine C5a does not require the COOH-terminal arginyl residue for spasmogenic activity. Further degradation of porcine des-Arg
74
-C5a by carboxypeptidase Y removed glycine-73 and leucine-72 and decreased the intrinsic spasmogenic activity by >90%. Hence, we conclude that, although the arginyl residue is not essential, the COOH-terminal sequence Leu-Gly-Arg contributes structural information that accounts for >90% of C5a activity. Human des-Arg
74
-C5a, like its porcine counterpart, has instrinsic anaphylatoxin activity; however, higher concentrations were needed to contract the guinea pig ileal tissue (i.e., 1 μM for human des-Arg
74
-C5a versus 1 nM for porcine des-Arg
74
-C5a). Furthermore, the des-Arg form of human C5a was only 0.1% as active as porcine des-Arg
74
-C5a for enhancing vascular permeability in guinea pig skin. In addition to these biological differences, numerous chemical differences exist between the human and porcine des-Arg
74
-C5a molecules, the most prominent feature being an oligosaccharide entity associated uniquely with the human C5a. When the oligosaccharide unit of human des-Arg
74
-C5a was removed by glycosidases, leaving a single glucosamine residue attached to the side chain of asparagine-64, activity was enhanced. The human des-Arg
74
-C5a molecule devoid of the complex oligosaccharide unit exhibited 10-fold stronger spasmogenic activity and 20- to 50-fold greater permeability-enhancing activity than did human des-Arg
74
-C5a containing the oligosaccharide. Consequently, the oligosaccharide associated with human C5a modulates or suppresses potentially harmful activities of this anaphylatoxin. The relatively high levels of spasmogenic activity associated with porcine des-Arg
74
-C5a indicate that this factor is poorly controlled by endogenous serum carboxypeptidase, whereas human C5a is virtually inactivated by the enzyme. Hence, the influence of this oligosaccharide in suppressing human des-Arg
74
-C5a activity is of major physiologic importance in protecting man from potentially toxic effects of this complement factor.
T cells respond preferentially to antigens that are similar to self H-2.
Authors: Bevan M J , Hünig T
Published: May 2006

We have constructed bone marrow irradiation chimeras to investigate the influence of self antigens on the specificity of the T lymphocyte receptor repertoire. Bone marrow cells from (A X B)F1 mice heterozygous for the major histocompatibility genes were allowed to mature into T cells in irradiated parent A or parent B strains. More than 8 weeks after irradiation, when the lymphoid system had regenerated from the F1 stem cells, the degree of T cell reactivity to mutant major histocompatibility antigens, A', was assessed. It was found that T cells that had matured in the irradiated A mice, [F1 leads to A] chimeras, responded better to A' antigen than did T cells from the [F1 leads to B] chimeras. Because the mutant histocompatibility antigen A' is very similar in structure to A, differing only by one or a few residues, this suggests that the T cell repertoire in [F1 leads to parent] chimeras reacts preferentially with foreign antigens that are slight variants of the self antigens expressed on radiation-resistant cells--probably cells in the thymus.
Independent recombination between avian leukosis virus terminal sequences and host DNA in virus-induced proliferative disease.
Authors: Neiman P , Beemon K , Luce J A
Published: May 2006

A cDNA transcript of Rous sarcoma virus, which contained the long terminal repeat (LTR) and some additional 3'-terminal sequences, was inserted into the plasmid pBR322. This recombinant plasmid, p53, was then used as a hybridization probe to detect viral terminal sequences in DNA from a number of tissues of birds with a variety of avian leukosis virus (ALV)-induced proliferative diseases. Using restriction endonuclease digestion and blot hybridization analysis, we detected, in addition to standard ALV genomes, viral terminal sequences linked to host DNA and not to viral genes. In DNA from bursal lymphomas and nephroblastomas, we observed small numbers of integration sites occupied by sequences in p53 and lacking most or all of the remainder of the viral genome. In DNA from osteopetrosis, we observed apparently multiple copies of molecules containing host DNA linked to viral LTR sequences. Some of these structures were contained in discrete, probably unintegrated, DNA molecules. We concluded that viral LTR sequences can be inserted as independent elements during recombination with host DNA in some forms of interaction between exogenous retroviruses and host cells. Because the LTRs have been implicated in integration and transcription of viral genes, the possibility that translocation or activation, or both, of host genes may occur as a consequence of viral infection is reinforced by these observations.
Identification and localization of a gene that specifies production of Escherichia coli DNA topoisomerase I.
Authors: Trucksis M , Depew R E
Published: May 2006

A gene that specifies production of Escherichia coli DNA topoisomerase I (omega protein) was identified with the aid of a radioimmunoassay for this protein. E. coli DNA topoisomerase I was produced by Salmonella typhimurium merodiploids that harbored E. coli plasmid F' 123, but not by strains that lost this plasmid. Analysis of strains with spontaneous deletions of F' 123 showed that the gene, topA, required for production of the E. coli omega protein was between the trp operon and the cysB gene. Deletions that eliminated topA also eliminated the supX gene. We suggest that topA is the structural gene of E. coli DNA topoisomerase I and that topA is identical to supX.
Mouse beta 2-microglobulin cDNA clones: a screening procedure for cDNA clones corresponding to rare mRNAs.
Authors: Parnes J R , Velan B , Felsenfeld A , Ramanathan L , Ferrini U , Appella E , Seidman J G
Published: May 2006

We have isolated three cDNA clones for beta 2-microglobulin, the small subunit of the major histocompatibility antigens. beta 2-Microglobulin makes up less than 0.1% of mouse liver protein, and its mRNA is approximately 0.03% of liver poly(A)+ mRNA. The cDNA clones were identified by screening 1400 cDNA clones made from 9--10S mouse liver poly(A)+ mRNA. The procedure for screening the cDNA clones involved binding pooled plasmid DNA to nitrocellulose filters and testing the ability of each filter to select beta 2-microglobulin mRNA. The filter-selected mRNAs were assayed for their ability to direct the synthesis of beta 2-microglobulin in translation reactions in vitro. The isolated clones were shown by nucleotide sequence analysis to encode beta 2-microglobulin. The positive-selection--hybridization assay has been modified to facilitate the screening of large numbers of cDNA clones, and the modified assay should allow the isolation of cDNAs corresponding to any mRNA whose in vitro translation products can be immunoprecipitated. These modifications are of particular value in the isolation of cDNA clones corresponding to rare species of mRNA.
Constancy of somatic DNA organization in developmentally regulated regions of the Drosophila genome.
Authors: Levine M , Garen A , Lepesant J A , Lepesant-Kejzlarova J
Published: May 2006

The purpose of this study was to test for the occurrence of changes in the organization, modification, or selective amplification of six developmentally regulated regions of genomic DNA during Drosophila development. Five of the regions contain structural genes, each of which maps at a single chromosomal site; the sixth region contains a dispersed segment that maps at about 30 different sites and appears to be transposable. The RNA transcripts and encoded proteins of the structural genes are major components of the fat body tissue in late third-instar larvae, in contrast to other larval tissues and young embryos in which the transcripts and proteins are hardly detectable. The dispersed segment shows the reverse developmental regulation: the amount of transcript is relatively high in young embryos and low in larval fat bodies. The test for changes in genomic DNA associated with the regulated expression of the six regions was based on comparative restriction mapping of the DNA from these sources. Genomic clones containing the transcribed and also flanking regions of DNA were used as probes to determine the positions of the complementary restriction fragments after electrophoresis in agarose gels. Each test, which involved digesting the genomic DNA samples with one of the endonucleases and probing with one of the clones, produced identical restriction maps for the different samples. The tests are capable of detecting changes in the organization of the cloned regions resulting from addition or removal either of endonuclease cleavage sites or of segments of DNA located between cleavage sites. Because the activities of several of the endonucleases, and probably all, are sensitive to methylation of one of the bases in the DNA recognition sequence, certain modifications of the cloned regions by methylation could also be detected. Although it is not certain that genomic reorganization or modification would have been detected by these tests, the number of endonucleases used was sufficient to provide persuasive evidence that such changes did not occur. Furthermore, the quantitative as well as qualitative similarities of the restriction maps in each test indicated that there was no significant selective amplification of the DNA within the cloned regions.
Coupling of protein antigens to erythrocytes through disulfide bond formation: preparation of stable and sensitive target cells for immune hemolysis.
Authors: Jou Y H , Bankert R B
Published: May 2006

An efficient technique has been developed for coupling protein antigens to erythrocyte membranes. The procedure involves three steps. First, 3-(2-pyridyldithio)propionyl residues are introduced into the protein by reaction with a heterobifunctional reagent, N-succinimidyl 3-(pyridyldithio) propionate. Second, the addition of disulfide groups to sheep erythrocytes (SRBC) is achieved by coupling dithiodiglycolic acid to SRBC with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The disulfide bonds of the dithiodiglycolyl-SRBC conjugate are then reduced with dithiothreitol. Finally, the 3-(2-pyridyldithio)propionyl-protein conjugate is covalently coupled to the thiolated SRBC through thiol/disulfide exchange to form the disulfide-linked antigen-SRBC conjugate. The procedure requires only 10-500 microgram of protein antigen for the preparation of 50 microliter of packed protein-coupled SRBC. Antibodies binding to antigen on the erythrocyte initiate a complement-dependent immune lysis of the target cells. Target cells prepared by this method are stable for at least 4 wk at 4 degrees C in phosphate buffer (pH 7.2) and are capable of detecting as little as 40 pg of antibody in a hemolytic assay without noticeable nonspecific lysis.
Prevention of 3-methylcholanthrene-induced fibrosarcomas in rats pre-inoculated with endogenous rat retrovirus.
Authors: Fish D C , Demarais J T , Djurickovic D B , Huebner R J
Published: May 2006

Weanling Fischer 344 rats received a single intraperitoneal injection of a 1000-fold concentrated preparation of endogenous nontransforming rat retrovirus. Ten days later, the rats were each given a single subcutaneous injection of 3-methylcholanthrene. The rats inoculated with the endogenous rat retrovirus were significantly protected against the development of cancer, whereas uninoculated rats and rats given one of several murine retroviruses or baboon retrovirus were not protected.
Stimulation of glucose incorporation and amino acid transport by insulin and an insulin-like growth factor in fibroblasts with defective insulin receptors cultured from a patient with leprechaunism.
Authors: Knight A B , Rechler M M , Romanus J A , Van Obberghen-Schilling E E , Nissley S P
Published: May 2006

Fibroblasts cultured from an infant with leprechaunism and insulin resistance have been reported to exhibit profound, selective defect in insulin binding. We now examine the effect of this defect on two acute metabolic actions of insulin thought to be mediated by the insulin receptor, glucose incorporation and N-methyl-alpha-aminoisobutyric acid (Me-AiBu) uptake. In the patient's fibroblasts, maximal insulin-stimulated glucose incorporation was less than 25% of that in control fibroblasts, whereas stimulation by hydrogen peroxide, an insulinomimetic agent that acts distal to the insulin receptor, was normal. By contrast, insulin stimulated Me-AiBu uptake to the same extent in patient's and control fibroblasts. Impaired glucose incorporation and relatively normal Me-AiBu uptake also were observed in the patient's cells with multiplication-stimulating activity, an insulin-like growth factor, despite the fact that multiplication-stimulating activity appeared to stimulate both responses in normal fibroblasts via an insulin-like growth factor receptor. The divergent effects on two hormone-stimulated functions in the patient's cells suggests differences in the coupling of a receptor to different effectors. The same coupling mechanisms appear to be used by insulin receptors and receptors for insulin-like growth factors.
Expression of the denV gene of bacteriophage T4 cloned in Escherichia coli.
Authors: Lloyd R S , Hanawalt P C
Published: May 2006

The denV gene of bacteriophage T4 has been cloned into Escherichia coli K-12 by inserting appropriate fragments of cytosine-containing T4 DNA into the Sal I site of the plasmid pBR322. The denV gene codes for an enzyme that initiates the excision repair of pyrimidine dimers produced in DNA by UV. In uvrA recA mutants, deficient in an early step in excision repair, the cloned DNA results in enhanced UV resistance that is more pronounced in stationary- than in exponential-phase cultures. The expression of the cloned DNA also results in the enhanced survival of UV-irradiated phage lambda or of a denV mutant of phage T4 and in removal of dimers from the DNA of UV-irradiated cells.
Determination of cellular RNA concentrations by electron microscopy of R loop-containing DNA.
Authors: Kaback D B , Rosbash M , Davidson N
Published: May 2006

R loop hybridizations and electron microscopy have been used to determine cellular RNA concentrations for cloned genes. In plasmid DNA sequence excess, all the complementary RNA is driven into R loop structures that can be assayed by electron microscopy. To determine the concentration of a particular poly(A)+ RNA, plasmid DNA crosslinked once every 2000-5000 base pairs with trioxsalen and UV light is hybridized in DNA sequence excess to various known amounts of total poly(A)+ RNA, and the R loops are stabilized by treatment with glyoxal. If necessary, excess nonhybridized RNA is removed by Sepharose 2B chromatography, which enables the visualization of less abundant transcripts. Reconstruction experiments demonstrated that electron microscopic determination of the fraction of plasmid DNA molecules containing specific RNA loops gives accurate values of specific RNA weight fractions or concentrations in the total poly(A)+ RNA populations. These methods were also used to determine the concentrations of five RNA species complementary to sequences on TRT3, a recombinant DNA plasmid containing yeast histone 2A and 2B genes and three other nonhistone genes. The methods described allow one to visualize the R loop structures for both abundant and nonabundant transcripts and to estimate concentrations of these RNA species simply by determining the fraction of DNA containing R loops.
Orientation of histidine residues in RNase A: neutron diffraction study.
Authors: Wlodawer A , Sjölin L
Published: May 2006

Difference Fourier maps have been calculated at 2.8-A resolution by using neutron diffraction data obtained from a single crystal of RNase A. The phases were derived from a model resulting from the joint refinement of x-ray and neutron data at 2.0-A and 2.8-A resolution, respectively. The orientation of histidine-48 assumed during the refinement of the x-ray model at 2.5 A was confirmed, whereas the other three histidines had to be rotated around C beta--C gamma bonds in order to agree with the neutron difference Fourier maps. In the final model, histidine-12 is clearly hydrogen bonded to the carbonyl oxygen of threonine-45 and to the oxygen of the inorganic phosphate, and histidine-119 is bonded to another oxygen of the phosphate and to the oxygen OD1 of aspartic acid-121.