bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of sarcomas in athymic mice.
Authors: Beattie G M , Knowles A F , Jensen F C , Baird S M , Kaplan N O
Published: May 2006

During the course of serial passage of 50 human xenografts in the athymic mouse over a period of 5 years we have observed two cases of induction of sarcomas in the murine stromal tissue associated with the human xenografts. Both times the growth of the murine sarcomas overtook that of the human xenograft. This change was monitored by analysis of the lactate dehydrogenase isozyme profile and histology of each passage of the human xenografts in the athymic mice. The two murine sarcomas were subsequently established in tissue culture. The sarcoma cell lines were found to be malignant by morphological and growth characteristics and were tumorigenic. They contained large amounts of murine leukemia virus when assayed for reverse transcriptase activity by infection of mouse SC-1 cells and BALB/c and NIH Swiss fibroblasts with filtered supernates, and some type C virus particles were observed by electron microscopy in tumor tissues. However, we were unable to demonstrate the presence of murine sarcoma virus by in vitro transformation of fibroblasts or sarcoma formation in vivo will cell free filtrates. Preliminary biochemical data indicate that the sarcomas are extremely high in plasma membrane ATP phosphohydrolase.
Ascorbate-quinone interactions: electrochemical, free radical, and cytotoxic properties.
Authors: Pethig R , Gascoyne P R , McLaughlin J A , Szent-Györgyi A
Published: May 2006

Standard midpoint potentials have been determined for p-benzoquinone, methoxy-p-benzoquinone and 2,3-, 2,5-, and 2,6-dimethoxy-p-benzoquinones in aqueous solution. ESR studies have been made of the ascorbate and semiquinone radicals produced when these quinones interact with sodium ascorbate. Direct correlations are found between the electrochemical potentials, generated semiquinone lifetimes, and cytotoxic action in Ehrlich ascites-bearing mice.
20-Hydroxyecdysone stimulates the accumulation of translatable yolk polypeptide gene transcript in adult male Drosophila melanogaster.
Authors: Shirk P D , Minoo P , Postlethwait J H
Published: May 2006

Yolk polypeptide (YP) synthesis is hormonally stimulated during maturation of adult female Drosophila melanogaster. Synthesis of the three YPs is sex specific and occurs in fat body cells and follicle cells of adult females. However, males have been shown to produce YPs when treated with the steroid hormone 20-hydroxyecdysone (20-HE). By using a cell-free translation system as an assay for YP mRNA, we found that 20-HE also causes the accumulation of translatable YP message in males. In addition, hybridization of cloned copies of genes for both YP1 and YP3 to total RNA from males showed that 20-HE caused the appearance of YP gene transcripts in males. Eight hours after treatment of males with 20-HE, YP gene transcript levels had increased at least 25-fold to approximately 2.7 x 10(6) copies of YP1 gene transcript per adult male fly. In normal adult females, there were 42 x 10(6) copies per fly by 24 hr. There was neither detectable YP synthesis nor translatable YP gene transcript in either normal 1- to 3-day-old males or 24-hr-old males treated with a juvenile hormone analogue. This evidence shows that 20-HE acts to regulate the levels of translatable YP mRNA in male Drosophila.
1 alpha,25-Dihydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3 prolong survival time of mice inoculated with myeloid leukemia cells.
Authors: Honma Y , Hozumi M , Abe E , Konno K , Fukushima M , Hata S , Nishii Y , DeLuca H F , Suda T
Published: May 2006

Syngeneic SL mice inoculated with murine myeloid leukemia cells (M1) all died of leukemia within 30 days. Treatment three times a week with 12.5-50 pmol per mouse of either 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], the active form of vitamin D3, or its synthetic analog, 1 alpha-hydroxyvitamin D3 [1 alpha(OH)D3], considerably prolonged the survival time of mice inoculated with M1 cells. 1 alpha(OH)D3 was more effective than 1 alpha,25(OH)2D3 in increasing the survival time of the mice. 1 alpha(OH)D3 also increased the survival time of nude mice inoculated with M1 cells. The 1 alpha(OH)[3H]D3 administered intraperitoneally to tumor-bearing mice was converted very rapidly to 1 alpha,25(OH)2-[3H]D3. The chronic administration of 25 pmol of 1 alpha(OH)D3 to tumor-bearing mice for 30 days caused no appreciable hypercalcemia. These results indicate clearly that 1 alpha,25(OH)2D3 is effective not only in inducing differentiation of M1 cells in vitro, as previously reported [Abe, E., Miyaura, C., Sakagami, H., Takeda, M., Konno, K., Yamazaki, T., Yoshiki, S. & Suda, T. (1981) Proc. Natl. Acad. Sci. USA 78, 4990-4994], but also in prolonging the survival time of mice inoculated with M1 cells.
Chromosomal assignment of the gene for the human beta 2-adrenergic receptor.
Authors: Sheppard J R , Wehner J M , McSwigan J D , Shows T B
Published: May 2006

Chinese hamster fibroblasts (CHW) fail to physiologically respond to the beta-adrenergic agonist isoproterenol with an increase in cellular cAMP. This unresponsiveness is due to a lack of beta-adrenergic receptors as indicated by an absence of specific binding of 125I-labeled hydroxybenzylpindolol (125I-HYP) to CHW plasma membranes. Preparation of somatic cell hybrids between CHW and human peripheral blood leukocytes led to the selection of a panel of 15 human-Chinese hamster cell hybrid clones, some of which had beta 2-adrenergic receptors (specifically bound 125I-HYP) and responded to isoproterenol (accumulated cAMP). Biochemical analysis of independent cloned cell hybrids indicated that beta 2-adrenergic receptor density and the intensity of the associated physiological response were closely correlated (r = 0.98). Both of these parameters were concordant in all cell hybrids with the presence of human chromosome 5. All other human chromosomes could be ruled out. These results suggest that the structural gene for the beta 2-adrenergic receptor is found on human chromosome 5.
Spontaneous H-2 mutants provide evidence that a copy mechanism analogous to gene conversion generates polymorphism in the major histocompatibility complex.
Authors: Pease L R , Schulze D H , Pfaffenbach G M , Nathenson S G
Published: May 2006

The analysis of H-2K products from spontaneously generated major histocompatibility complex (MHC) mutants and of the primary structure of other class I antigens suggests the genetic hypothesis that diversity in the MHC results from a copy mechanism analogous to gene conversion. The hypothesis was tested by making precise structural predictions about three partially characterized MHC mutants (bm1, bm3, and bm8). The predictions were based on consensus sequences among class I genes that differ from H-2Kb in the same region of the molecule as do the Kb mutants. In two cases (bm3 and bm8) we successfully predicted the correct amino acid substitution at positions known to be altered but for which the specific nature of the substitution had not been determined. In two additional cases (bm1 and bm8) we predicted and found both new mutation sites and the specific amino acid substitutions. The positions and identifications of the variant amino acids were determined by radiolabeled amino acid sequence analysis and DNA restriction endonuclease analysis. The interaction of MHC genes through a copy mechanism to generate diversity permits the introduction of multiple nucleotide base substitutions into class I sequences by a single genetic event. Such a mechanism may account in part for the large structural divergence among alleles of MHC loci and the high degree of MHC polymorphism among wild mice.
Cloning and expression of DNA sequences associated with the killer trait of Paramecium tetraurelia stock 47.
Authors: Quackenbush R L , Burbach J A
Published: May 2006

We have presented direct evidence that at least one of the traits associated with killing of paramecia by kappa particles is determined by an extrachromosomal genetic element. Plasmid DNA was isolated from Caedibacter taeniospiralis 47 (commonly known as 47 kappa), which is an obligate cytoplasmic endosymbiont of Paramecium tetraurelia. Fragments of pKAP47 DNA generated by Pst I digestion were inserted into pBR328 and then introduced into Escherichia coli 294 by transformation. Clones carrying recombinant plasmids were screened for toxicity toward sensitive strains of paramecia or for the ability to produce R bodies. None of the clones appeared to be toxic. However, three clones were found to have the ability to produce R bodies, which are proteinaceous ribbons (10-20 microns long, 0.5 microns wide, and 13 nm thick) rolled up inside the cell to form a hollow cylinder about 0.5 microns in diameter and 0.5 microns long. Each of these clones carry plasmids that contain the Pst I B fragment from pKAP47. Subclones of one of the recombinant plasmids, pBQ51, were constructed to determine the approximate location of DNA sequences necessary for R-body synthesis. The left-hand boundary of the required sequences was found to occur within a 600-base-pair region, and the location of the right-hand boundary was determined to occur within a 700-base-pair region. The minimum and maximum sizes of sequences required for R-body synthesis are between 1,300 and 2,600 base pairs.
Thermodynamic efficiency of microbial growth is low but optimal for maximal growth rate
Authors: Westerhoff Hans V. , Hellingwerf Klaas J. , Van Dam Karel
Published: May 2006

Thermodynamic efficiency of microbial growth on substrates that are more oxidized than biomass approaches 24%. This is the theoretical value for a linear energy converter optimized for maximal output flow at optimal efficiency. For growth on substrates more reduced than biomass, thermodynamic efficiencies correspond to those predicted for optimization to maximal growth rate (or yield) only.
Appearance of a cytosolic protein that stimulates glyceraldehyde-3-phosphate dehydrogenase activity during initiation of renal epithelial cell growth.
Authors: Aithal H N , Walsh-Reitz M M , Toback F G
Published: May 2006

Rats fed a K-deficient diet exhibit accelerated kidney growth and enhanced activity of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD; D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase, EC 1.2.1.12). Cultures of renal epithelial cells (BSC-1 line) served as a model for this phenomenon because a reduction in the medium K concentration from 5.4 to 3.2 mM resulted in augmented growth and increased G3PD activity. Mixing the soluble supernatant fraction from cells grown in low-K medium (3.2 mM) with that from cells grown in control medium indicated that the cytosol of low-K cells contained a positive modifier of G3PD activity. Appearance of modifier activity that occurred within 1 hr after exposure of cells to low-K medium was blocked by cycloheximide but not by actinomycin D. Modifier activity was also observed in mouse fibroblasts stimulated by low-K medium and in proliferating renal medullary tissue from rats fed a K-deficient diet. A single protein that contained G3PD-stimulating activity was isolated from the soluble supernatant of BSC-1 cells exposed to low-K medium. This protein was not detectable in control cells. The material yielded a single band on NaDodSO4/polyacrylamide gel electrophoresis with an apparent Mr of 62,000. These results suggest that a new protein with the capacity to stimulate G3PD activity appears in the cytosol during the initiation of cell growth.
When does the good of the group override the advantage of the individual?
Authors: Leigh Egbert Giles
Published: May 2006

In a system of
N
populations of
n
reproductive individuals apiece, in which each population has constant variance
v
2
and lasts
L
generations, group selection on a quantitative character has a reasonable chance of overriding selection within populations if (and only if) the populations never exchange migrants, each population is founded by colonists from a single parent population, and the number of populations exceeds the effective number of reproductive individuals per population. If each population derives from a single parent population, then the exchange of a single successful migrant per population per
L
generations can triple the strength of group selection required to overcome a given selection within populations. If populations exchange no migrants, then the derivation of one in every
N
populations from two equally represented parents (while the others all derive from a single parent) doubles the strength of group selection required to prevail. Group selection is accordingly likely to be effective only in certain categories of parasites.
Lack of metabolism as the biochemical basis of bleomycin-induced pulmonary toxicity.
Authors: Lazo J S , Humphreys C J
Published: May 2006

The biochemical basis for bleomycin-induced pulmonary toxicity was studied in vitro and in vivo with an improved HPLC system. The in vitro metabolism of bleomycin A2 to desamido-bleomycin A2 was measured in tissue homogenates from a species sensitive (mice) and relatively resistant (rabbits) to the pulmonary fibrogenic properties of bleomycin. Lung tissue from mice lacked detectable bleomycin hydrolase activity, whereas rabbit lung tissue homogenates had high levels of the enzyme activity, equaling that seen in rabbit kidneys and spleen. Injection of radiolabeled bleomycin A2 into mice demonstrated that only a small percentage of the total dose was taken up by any organ and that extensive metabolism of this drug occurred within 1 hr in liver, kidneys, and spleen but not in lungs in vivo. In addition, metabolites other than desamido-bleomycin A2 were prominent, and their relative amounts increased with time. Mice injected subcutaneously with bleomycin A2 developed pulmonary fibrosis, while animals treated with equivalent doses of desamido-bleomycin A2 did not, indicating that this metabolite is not as toxic to the lungs as is the parent compound. These results provide direct evidence that metabolism plays a major role in determining the toxic potential of bleomycin to the lungs.
Hemolytic activity of influenza virus hemagglutinin glycoproteins activated in mildly acidic environments.
Authors: Sato S B , Kawasaki K , Ohnishi S
Published: May 2006

Hemagglutinin (HA) glycoproteins isolated from influenza virus caused hemolysis and liposome lysis at pH less than 6.0. The pH dependence was similar to that of the parent virus. Hemagglutination and hemolysis titers of HA were comparable with those of virus. The time course of hemolysis by HA was somewhat different from that by virus. HA did not cause fusion of erythrocytes in acidic media, in contrast to virus. Both HA and virus, previously incubated at pH less than 6.0, lost their low-pH-induced hemolytic activity. Isolated HA formed rosette-like structures at neutral pH, and these aggregated in acidic media. Virus also aggregated in acidic media and its envelope became leaky to negative stain. HA previously incubated at pH less than 6.0 became susceptible to trypsin digestion. Both reversible and irreversible structural changes of HA were observed by fluorescence spectroscopy; a reversible change at a pH between neutral and 6.4 and an irreversible one at pH less than 6.0. Bromelain-released HA did not cause hemolysis and liposome lysis in acidic media. The precursor form of HA did not have hemolytic activity in acidic media. The similarity in pH dependence indicates that the structural change in HA induced at pH less than 6.0 is the cause of activation and inactivation of hemolysis, HA and virus aggregation, and trypsin susceptibility. We propose that the hydrophobic NH2-terminal segment of HA2 is exposed during the structural change and interacts with the target membranes, causing a permeability increase and leading to hemolysis and lysis. The virus-induced hemolysis can be ascribed for the most part to envelope fusion activated in acidic media.
Complete differentiation between enkephalinase and angiotensin-converting enzyme inhibition by retro-thiorphan.
Authors: Roques B P , Lucas-Soroca E , Chaillet P , Costentin J , Fournié-Zaluski M C
Published: May 2006

Thiorphan, N-[(R,S)-3-mercapto-2-benzylpropanoyl]glycine is a highly potent inhibitor (Ki = 3.5 nM) of "enkephalinase," a metalloendopeptidase cleaving the Gly-Phe bond (positions 3 and 4) of enkephalins in brain tissue. In accordance with this property, thiorphan displays antinociceptive activity after systemic administration. However, thiorphan also inhibits to a lesser extent (Ki = 140 nM) the widely distributed angiotensin-converting enzyme, a carboxydipeptidase implicated in blood pressure regulation. Therefore, in view of an eventual clinical use of enkephalinase inhibitors, it was very important to develop fully specific compounds. Such derivatives were obtained taking into account that N-methylation of the ultimate amide bond of dipeptides strongly decreases enkephalinase affinity without affecting angiotension-converting enzyme recognition, whereas retro-inversion of the amide bond leads to the inverse effect. Thus, the retro-inverso dipeptide (R)-H2N-CH(CH2 phi)-NHCO-CH2-CO2H exhibits an inhibitory potency on enkephalinase (IC50 approximately equal to 12 muM) close to that of the natural dipeptide L-Phe-Gly (IC50 approximately equal to 3 muM). This result shows the topological analogy between the crucial components involved in enkephalinase recognition both in active dipeptides and structurally related retro-inverso isomers. Taking into account these observations, retro-thiorphan, (R,S)-HS-CH2-CH-(CH2 phi)-NHCO-CH2-COOH, was prepared. As compared to thiorphan, the retro isomer is 50% as potent (Ki = 6 nM) on enkephalinase but displays a drastic loss of potency on angiotension-converting enzyme (IC50 greater than 10,000 nM). This specificity was interpreted as a consequence of differences in the stereochemical constraints involving enzyme-inhibitor hydrogen bonding. This hypothesis is supported by reported crystallographic studies on related enzymes such as thermolysin and carboxypeptidase A. As expected, retro-thiorphan exhibits about the same analgesic potency as thiorphan on the hot plate and writhing tests in mice. Therefore, the topological concept of retro-inverso isomers could be extended to other enkephalinase inhibitors, allowing the design of potent and highly selective compounds occurring as new classes of analgesic and psychoactive agents.
Close relationship between the long terminal repeats of avian leukosis-sarcoma virus and copia-like movable genetic elements of Drosophila.
Authors: Kugimiya W , Ikenaga H , Saigo K
Published: May 2006

A new species of copia-like movable genetic element termed 17.6 was identified in Drosophila melanogaster, and the nucleotide sequences of its long terminal repeats (LTRs) were determined. The LTRs of 17.6 were not only homologous to those of 297, a sibling movable genetic element of 17.6, but also closely matched those of avian leukosis-sarcoma virus. This made it possible (i) to identify the nucleotide sequences in 17.6 and 297 that correspond to the crucial regulatory sequences for both transcription and reverse transcription in avian leukosis-sarcoma virus and (ii) to divide the LTRs of these two elements into three regions, U3, R, and U5, like those of retrovirus proviruses. Similarity in sequence was also found to a certain extent in other copia-like elements. From these results, we postulate that copia-like movable genetic elements in Drosophila originated from infection of a progenitor Drosophila with a retrovirus from which the present-day avian leukosis-sarcoma virus was derived.
Suggested model for prebiotic evolution: the use of chaos.
Authors: Anderson P W
Published: May 2006

I elaborate on an earlier model for prebiotic evolution by adding a selection function depending randomly on base-pair correlations along the replicating RNA chains. By analogy with the equilibrium statistics of spin glass, this should give a wide variety of metastable "species" that can mutate or combine symbiotically.
The primitive code and repeats of base oligomers as the primordial protein-encoding sequence.
Authors: Ohno S , Epplen J T
Published: May 2006

Even if the prebiotic self-replication of nucleic acids and the subsequent emergence of primitive, enzyme-independent tRNAs are accepted as plausible, the origin of life by spontaneous generation still appears improbable. This is because the just-emerged primitive translational machinery had to cope with base sequences that were not preselected for their coding potentials. Particularly if the primitive mitochondria-like code with four chain-terminating base triplets preceded the universal code, the translation of long, randomly generated, base sequences at this critical stage would have merely resulted in the production of short oligopeptides instead of long polypeptide chains. We present the base sequence of a mouse transcript containing tetranucleotide repeats conserved during evolution. Even if translated in accordance with the primitive mitochondria-like code, this transcript in its three reading frames can yield 245-, 246-, and 251-residue-long tetrapeptidic periodical polypeptides that are already acquiring longer periodicities. We contend that the first set of base sequences translated at the beginning of life were such oligonucleotide repeats. By quickly acquiring longer periodicities, their products must have soon gained characteristic secondary structures--alpha-helical or beta-sheet or both.
Fine structure analysis and nucleotide sequence of the vaccinia virus thymidine kinase gene.
Authors: Hruby D E , Maki R A , Miller D B , Ball L A
Published: May 2006

The thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene of vaccinia virus has previously been mapped near the middle of the viral DNA, within the 4.85-kilobase HindIII J fragment, and shown to encode a Mr 19,000 polypeptide [Hruby, D. E. & Ball, L. A. (1982) J. Virol. 43, 403-409]. To locate the gene more precisely and to determine the structure of the basic transcriptional unit, the positions of cleavage sites for several restriction endonucleases were mapped within the HindIII J DNA fragment. Four appropriate subfragments of HindIII J DNA were inserted into plasmid pBR322 derivatives and cloned in Escherichia coli. These recombinant plasmid DNAs were tested for their ability to inhibit the cell-free synthesis of active thymidine kinase and to retain the mRNA for this enzyme when immobilized on nitrocellulose filters. The data showed that the gene spanned an EcoRI cleavage site that lies 850 base pairs from the left-hand end of the HindIII J fragment (the HindIII L-J boundary). Because hybridization of vaccinia virus DNA to partially purified thymidine kinase mRNA detected only a single 670-nucleotide RNA species capable of hybridizing to this region of the genome, nuclease S1 mapping experiments were carried out with thymidine kinase mRNA to protect DNA fragments that were terminally labeled at this EcoRI site. The results indicated that the gene extended from about 550 to 1,150 base pairs from the left end of HindIII J, was transcribed in a rightward direction, and contained no intervening sequences. Hence, a 1.04-kilobase Ava II-Hpa II restriction fragment containing this region of DNA was isolated and subjected to nucleotide sequence analysis. An examination of this nucleotide sequence revealed the presence of an open reading frame of 531 nucleotides capable of encoding a protein of 177 amino acids with a Mr of 20,077.
Dynamics of a small globular protein in terms of low-frequency vibrational modes.
Authors: Go N , Noguti T , Nishikawa T
Published: May 2006

Normal modes of low-frequency vibrations are calculated for a small globular protein, bovine pancreatic trypsin inhibitor. In modes with frequencies below 120 cm-1 the protein molecule behaves like a continuous elastic body. Most modes with frequencies above 50 cm-1 are shown to behave harmonically within the range of thermal fluctuations at room temperature. Those with frequencies below 50 cm-1 show some anharmonicity. Magnitudes of displacements of atoms are mainly determined by the modes with frequencies below 30 cm-1. These very-low-frequency modes contribute significantly to the entropy of the system. The dynamic structure of the globular protein is described as a superposition of harmonic high-frequency motions and coupled anharmonic low-frequency motions of collective variables corresponding to the normal modes of vibration.
Analysis of two-dimensional protein patterns from mouse embryos with different trisomies.
Authors: Klose J , Putz B
Published: May 2006

Two-dimensional protein patterns from whole mouse embryos with different trisomies (Ts) (Ts1, -12, -14, -19) and from different organs (normal or malformed) and developmental stages of Ts12 embryos were analyzed by comparison with control patterns. Quantitatively altered proteins were found, and a portion of these (21/approximately equal to 1,000, average) was attributable to the Ts conditions. Most of these variants were found always (regularly) in Ts embryos. They could be divided into two groups: group I shows characteristics (chromosome specific, density increased by a factor close to 1.5 +/- 0.12) compatible with proteins directly affected by the Ts; and group II (chromosome nonspecific, density decreased, seldom increased) results most likely from indirect effects. The incidence of group II variants (about 13/approximately equal to 1,000, average) was considerably greater than that of the group I variants (about 3/approximately equal to 1,000). The frequency of both types of variants, however, was far lower than was expected by a rough estimation. Apparently, a relatively small number of changes, rather than a complex, escalating effect, was induced at the protein level by the Ts. Some observations suggest that this is due to a stable regulation of protein concentrations. The proportion in which quantitative changes of different types occurred in the protein patterns did not correlate with the degree of developmental impairments (malformation, retardation, early death) of the embryos. The generalized occurrence of protein changes on the cellular level might explain the restricted viability of Ts mouse embryos.
Iron superoxide dismutase from Escherichia coli at 3.1-A resolution: a structure unlike that of copper/zinc protein at both monomer and dimer levels.
Authors: Stallings W C , Powers T B , Pattridge K A , Fee J A , Ludwig M L
Published: May 2006

The structure of iron superoxide dismutase (EC 1.15.1.1) from Escherichia coli has been determined at 3.1-A resolution. The dimeric molecule is constructed from identical subunits, which are two-domain polypeptides. The NH2-terminal domain is composed of two antiparallel crossing helices and the COOH-terminal domain is a three-layered structure characterized by mixed alpha/beta secondary structural features. The active center iron atoms, separated by 18 A and located near the monomer-monomer interface, are coordinated by two amino acid residues from each domain. Azide binding has been investigated by using difference Fourier techniques. Consistent with the notion of the independent evolution of the copper/zinc dismutase gene, the iron dismutase structure resembles the copper/zinc protein at neither the monomer nor the dimer level.
Partial mRNA sequences for human A alpha, B beta, and gamma fibrinogen chains: evolutionary and functional implications.
Authors: Kant J A , Lord S T , Crabtree G R
Published: May 2006

Using rat cDNA and genomic probes to screen a human liver cDNA library, we have isolated clone of 2,274, 855, and 736 base pairs (bp) coding for the A alpha, B beta and gamma chains of human fibrinogen. Sequence analysis reveals a hitherto unrecognized extension of 15 amino acids at the carboxyl terminus of the A alpha chain, the terminal residue of which is proline. This brings the known length of the human A alpha chain to 625 amino acids. The 13-amino-acid repeated region in the midportion of the A alpha chain clearly has arisen through an 8-fold duplication of a 39-bp genetic element, which itself appears to have been constructed from smaller 6-bp repeating units. Greater than 50% sequence homology between B beta- and gamma-chain coding regions confirms postulates that these genes have arisen by duplication and subsequent divergence of an ancestral gene. A comparison of human and rat gamma-chain cDNAs shows more than 88% sequence homology over the carboxyl-terminal 162 amino acids, implying strong selective pressures on these portions of the gamma-chain gene.
Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination.
Authors: Biggin M D , Gibson T J , Hong G F
Published: May 2006

Two methods for increasing the length of DNA sequence data that can be read off a polyacrylamide gel are described. We have developed a rapid way to pour a buffer concentration gradient gel that, by altering the vertical band separation on an autoradiograph, allows more sequence to be obtained from a gel. We also show that the use of deoxyadenosine 5'-(alpha-[35S]thio)triphosphate as the label incorporated in dideoxynucleotide sequence reactions increases the sharpness of the bands on an autoradiograph and so increases the resolution achieved.
9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine: a selective inhibitor of herpes group virus replication.
Authors: Field A K , Davies M E , DeWitt C , Perry H C , Liou R , Germershausen J , Karkas J D , Ashton W T , Johnston D B , Tolman R L
Published: May 2006

9-([2-Hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (2'-nor-2'-deoxyguanosine; 2'NDG) selectively inhibits the replication of herpes group viruses. In cell culture studies 2'NDG was at least 10-fold more potent than acyclovir (ACV) in inhibition of human cytomegalovirus replication and Epstein-Barr virus-induced lymphocyte transformation and was about as effective as ACV in inhibition of herpes simplex viruses 1 and 2 and varicella zoster virus. Orally administered 2'NDG was 6- to 50-fold more efficacious than ACV in treating systemic or local HSV-1 infection or HSV-2 intravaginal infection in mice. The mode of action of 2'NDG appears to involve phosphorylation by herpes simplex virus thymidine kinase and subsequent phosphorylations by cellular kinases to produce 2'NDG triphosphate, which is a potent inhibitor of herpes virus DNA polymerase. Compared to ACV, 2'NDG was a more efficient substrate for HSV-1 thymidine kinase (Vmax/Km for 2'NDG 30-fold higher than that of ACV), whereas 2'NDG monophosphate is a more efficient substrate for GMP kinase (Vmax/Km for 2'NDG monophosphate 492-fold higher than that for ACV monophosphate). The combined effect is more rapid production of the inhibitory triphosphate from 2'NDG than from ACV.
Nucleolar dominance in polytene cells of
Drosophila
Authors: Endow Sharyn A.
Published: May 2006

Previous studies indicate that genes from only one of the cell's nucleolus organizers undergo multiple rounds of DNA replication in polytene cells of
Drosophila
. This report presents evidence that this effect is mediated by a function that is associated with the ribosomal genes of the dominant or replicating X chromosome. This function can act in
trans
to result in replication of the ribosomal genes on the recessive X chromosome in flies that are bobbed for the dominant X chromosome. In these cases, ribosomal genes from both chromosomes undergo polytenization. Heterochromatic regions that flank the nucleolus organizer have little or no effect on nucleolar dominance. In addition, deletion of the compensatory response (
cr
+
) locus does not affect the dominance, suggesting that ribosomal gene compensation and nucleolar dominance in polytene cells of
Drosophila
are separate genetic phenomena.
Expression of bacterial genes in plant cells.
Authors: Fraley R T , Rogers S G , Horsch R B , Sanders P R , Flick J S , Adams S P , Bittner M L , Brand L A , Fink C L , Fry J S , Galluppi G R , Goldberg S B , Hoffmann N L , Woo S C
Published: May 2006

Chimeric bacterial genes conferring resistance to aminoglycoside antibiotics have been inserted into the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid and introduced into plant cells by in vitro transformation techniques. The chimeric genes contain the nopaline synthase 5' and 3' regulatory regions joined to the genes for neomycin phosphotransferase type I or type II. The chimeric genes were cloned into an intermediate vector, pMON120, and inserted into pTiB6S3 by recombination and then introduced into petunia and tobacco cells by cocultivating A. tumefaciens cells with protoplast-derived cells. Southern hybridization was used to confirm the presence of the chimeric genes in the transformed plant tissues. Expression of the chimeric genes was determined by the ability of the transformed cells to proliferate on medium containing normally inhibitory levels of kanamycin (50 micrograms/ml) or other aminoglycoside antibiotics. Plant cells transformed by wild-type pTiB6S3 or derivatives carrying the bacterial neomycin phosphotransferase genes with their own promoters failed to grow under these conditions. The significance of these results for plant genetic engineering is discussed.
Reconstitution of beta-adrenergic receptors in lipid vesicles: affinity chromatography-purified receptors confer catecholamine responsiveness on a heterologous adenylate cyclase system.
Authors: Cerione R A , Strulovici B , Benovic J L , Strader C D , Caron M G , Lefkowitz R J
Published: May 2006

The binding function of purified receptors can be assessed with radioligands, but the interaction of receptors with their biochemical effectors has not been amenable to direct study. Toward this end, procedures have been developed for directly demonstrating functionality of purified beta-adrenergic receptor preparations. Digitonin-solubilized beta-adrenergic receptors from frog erythrocytes or rat lung were purified approximately equal to 100- to 5,000-fold by affinity chromatography and inserted into a mixture of frog erythrocyte lipids and dimyristoyl phosphatidylcholine in the presence of octyl glucoside. Reconstitution of beta-adrenergic receptor binding was typically 25-50% and could also be effected with soybean phosphatidylcholine in the presence of octyl glucoside. The reconstituted beta-adrenergic receptors were then fused with Xenopus laevis erythrocytes, which contain prostaglandin E1-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] but few beta-adrenergic receptors and little or no catecholamine-sensitive adenylate cyclase. Fusion of reconstituted receptor with Xenopus laevis erythrocytes establishes a substantial (2- to 10-fold) stimulation of the hybrid adenylate cyclase by the beta-agonist isoproterenol. The extent of stimulation depends on the amount of reconstituted beta-adrenergic receptor added, is blocked by propranolol, and is eliminated by boiling the beta-adrenergic receptor prior to reconstitution. The successful coupling of a purified receptor to a heterologous adenylate cyclase opens the way to the study of receptor structure---function relationships.
Methylation of replicating and post-replicated mouse L-cell DNA.
Authors: Gruenbaum Y , Szyf M , Cedar H , Razin A
Published: May 2006

We have introduced [alpha-32P]dGTP into permeabilized cells and measured the degree of methylation at CpG sites by nearest-neighbor analysis. This method reveals a lag of approximately 1 min between DNA synthesis and the modification event. When methylation is inhibited by the addition of S-adenosyl-L-homocysteine in the presence of continued DNA synthesis, the resulting hemimethylated sites are methylated immediately after the release of inhibition. The results suggest that the methylase activity in the cell allows immediate methylation but conditions at the replication fork bring about a short delay in the onset of the modification reaction.
12-fold difference between the critical monomer concentrations of the two ends of actin filaments in physiological salt conditions.
Authors: Wegner A , Isenberg G
Published: May 2006

We determined the critical monomer concentrations at which association and dissociation reactions are balanced at the two ends of actin filaments. For measurement of the critical concentration of the pointed end, interference with the high dynamics of the barbed end was excluded by capping the barbed ends with an actin filament capping protein isolated from bovine brain. The critical concentration of the pointed end (1.5 microM) was found to be 12- to 15-fold higher than the critical concentration of the barbed end (0.10-0.12 microM) at a temperature of 37 degrees C and physiological salt concentrations (100 mM KCl/1-2 mM MgCl2/0.3 mM EGTA or 0.2 mM CaCl2, pH 7.5).
Identification of glucocorticoid-induced genes in rat hepatoma cells by isolation of cloned cDNA sequences.
Authors: Feinberg R F , Sun L H , Ordahl C P , Frankel F R
Published: May 2006

The expression of specific cellular genes in M1.19 rat hepatoma cells involves glucocorticoid regulation by mechanisms that are not well understood. To approach this problem we cloned cDNA prepared from dexamethasone-induced poly(A)-RNA and used a comparative colony hybridization method to identify recombinant clones containing hormone-regulated sequences. Two such cDNA clones, p1394 and p255, hybridize to a homogeneous RNA species of 900 nucleotides that is present in high abundance in 24-hr-induced cells but is undetectable in uninduced cells. This RNA can be seen as early as 1 hr after dexamethasone stimulation. Inhibition of protein synthesis with cycloheximide significantly reduces the accumulation of the RNA but does not abolish the induction response. In normal adult rat liver the RNA is abundant, and this RNA is induced by dexamethasone in adrenalectomized rats. Plasmids p1394 and p255 contain sequences that are homologous to the mRNA coding for the acute-phase reactant protein alpha 1-acid glycoprotein. Two other cDNA clones, p655 and p333, hybridize to a more heterogeneous RNA species 200-400 nucleotides in size with a lower induction response to dexamethasone. Southern blot analysis of M1.19 genomic DNA indicates that p1394 and p255 are complementary to a single DNA fragment, whereas p655 and p333 are complementary to repetitive sequences in the M1.19 genome. It appears that the genetic domain of glucocorticoid control in M1.19 rat hepatoma cells involves low copy number genes such as alpha 1-acid glycoprotein as well as repetitive sequence elements.
Identification in pituitary tissue of a peptide alpha-amidation activity that acts on glycine-extended peptides and requires molecular oxygen, copper, and ascorbic acid.
Authors: Eipper B A , Mains R E , Glembotski C C
Published: May 2006

An enzymatic activity capable of producing an alpha-amidated peptide product from its glycine-extended precursor has been identified in secretory granules of rat anterior, intermediate, and neural pituitary and bovine intermediate pituitary. High levels of endogenous inhibitors of this alpha-amidation activity have also been found in tissue homogenates. The alpha-amidation activity is totally inhibited by addition of divalent metal ion chelators such as diethyldithiocarbamate, o-phenanthroline, and EDTA; alpha-amidation activity is restored to above control levels upon addition of copper. The alpha-amidation reaction requires the presence of molecular oxygen. Of the various cofactors tested, ascorbic acid was the most potent stimulator of alpha-amidation. The alpha-amidation activity has a neutral pH optimum and is primarily soluble following several cycles of freezing and thawing. Kinetic studies with the bovine intermediate pituitary granule-associated activity demonstrated a linear Lineweaver-Burk plot when D-Tyr-Val-Gly was the varied substrate; the apparent Km and Vmax varied with the concentration of ascorbic acid. The substrate specificity of the alpha-amidation activity appears to be quite broad; the conversion of D-Tyr-Val-Gly into D-Tyr-Val-NH2 is inhibited by the addition of a variety of glycine-extended peptides.
Versatile cosmid vectors for the isolation, expression, and rescue of gene sequences: studies with the human alpha-globin gene cluster.
Authors: Lau Y F , Kan Y W
Published: May 2006

We have developed a series of cosmids that can be used as vectors for genomic recombinant DNA library preparations, as expression vectors in mammalian cells for both transient and stable transformations, and as shuttle vectors between bacteria and mammalian cells. These cosmids were constructed by inserting one of the SV2-derived selectable gene markers--SV2-gpt, SV2-DHFR, and SV2-neo--in cosmid pJB8. High efficiency of genomic cloning was obtained with these cosmids and the size of the inserts was 30-42 kilobases. We isolated recombinant cosmids containing the human alpha-globin gene cluster from these genomic libraries. The simian virus 40 DNA in these selectable gene markers provides the origin of replication and enhancer sequences necessary for replication in permissive cells such as COS 7 cells and thereby allows transient expression of alpha-globin genes in these cells. These cosmids and their recombinants could also be stably transformed into mammalian cells by using the respective selection systems. Both of the adult alpha-globin genes were more actively expressed than the embryonic zeta-globin genes in these transformed cell lines. Because of the presence of the cohesive ends of the Charon 4A phage in the cosmids, the transforming DNA sequences could readily be rescued from these stably transformed cells into bacteria by in vitro packaging of total cellular DNA. Thus, these cosmid vectors are potentially useful for direct isolation of structural genes.
Calcium/calmodulin inhibition of coupled NaCl transport in membrane vesicles from rabbit ileal brush border.
Authors: Fan C C , Powell D W
Published: May 2006

The role of Ca2+ and calmodulin in regulating coupled NaCl transport has been investigated in membrane vesicles from rabbit ileal brush border. Uptake of 22Na+ and 36Cl- was determined by a rapid filtration technique in vesicles isolated with a sucrose density gradient ultracentrifugation method. Ca2+ on the inside of the vesicle inhibited Na+ uptake when Cl- was the anion and Cl- uptake when Na+ was the cation by approximately equal to 30%. Ca2+ on the outside had no effect. When gluconate was the anion or when choline was the cation, Na+ or Cl- uptake was reduced by only 9-12%. A similar inhibition of D-[3H]mannitol uptake (10-17%) suggests this was due to a nonspecific decrease in the membrane permeability. Other cations such as Ba2+ and Mg2+ had no effect, but La3+ inhibited Na+ and Cl- uptake to the same degree as Ca2+. Calmodulin (2 microM) in combination with Ca2+ (1 microM, free concentration) significantly inhibited Na+ uptake when Cl- was the anion by 21-32% and Cl- uptake when Na+ was the cation by 20-27%. This effect was completely reversed by 10 microM trifluoperazine. When gluconate was the anion or when choline was the cation, Na+ or Cl- uptake was unaffected by Ca2+/calmodulin and trifluoperazine. The Ki for Ca2+ inhibition of Cl- -coupled Na+ uptake was reduced from 200 microM to 0.2 microM by incubation with 20 microM calmodulin. The Ki for exogenously added calmodulin studied at 1 microM Ca2+ was 0.2 microM. The Ki for trifluoperazine inhibition of the Ca2+/calmodulin response was 3 microM. These results represent compelling evidence for intracellular Ca2+/calmodulin regulation of coupled NaCl transport across the intestinal microvillus membrane. The exact mechanism of this regulation remains to be delineated.
Identification of key regulated events early in the life of hybrid animal cells constructed by nuclear transplantation.
Authors: Hightower M J , Bruno J , Lucas J J
Published: May 2006

Reconstituted cells were constructed by fusion of cytoplasts from the human diploid fibroblast cell strain Detroit 532 and karyoplasts from the mouse fibroblast cell line A9. Several cellular properties were examined during the first 48 hr after nuclear transplantation. (i) The overall morphology of the cells originally resembled that of the cytoplasmic donor, Detroit 532, but rapidly changed to approximate that of the nuclear donor, A9. However, definitive changes in the microfilament structure of the reconstituted cells were not seen until 24-48 hr after fusion. These observations support the idea that the presence or absence of an ordered array of microfilament bundles is not the sole determinant of cell shape. (ii) Although cytoplasts and karyoplasts were prepared from cultures of randomly growing cells, the first division of reconstituted cells occurred in a synchronous manner. However, the initiation of DNA synthesis was not synchronized. It thus appeared that, in their first cell cycle, the cells had a G2 period of variable length. The results further suggest that the cytoplasm of interphase fibroblasts contains the material necessary to initiate or support DNA synthesis in a transplanted nucleus but not entry into mitosis. (iii) A two-dimensional gel electrophoretic analysis of polypeptide synthesis in reconstituted cell cultures showed that synthesis directed by transplanted mouse nuclei could be detected as early as 3-6 hr after fusion. Some of the mouse polypeptides detected at the earliest time points studied were not among the major polypeptides synthesized by the parental A9 cells. By about 48 hr after fusion, the pattern of polypeptides produced by reconstituted cells was almost indistinguishable from that of the nuclear donor parent cells.
Metabolite regulation of heat shock protein levels.
Authors: Lanks K W
Published: May 2006

When murine L929 cells are briefly exposed to elevated temperature (43 degrees C) they preferentially synthesize heat shock proteins (HSPs) of Mrs 85,000 and 69,000. By the criteria of two-dimensional polyacrylamide gel electrophoresis and partial proteolytic peptide mapping, the Mr 85,000 HSP is indistinguishable from the major cytoplasmic protein whose synthesis and intracellular level were shown previously to be suppressed by glucose deprivation. The mechanism regulating the Mr 85,000 HSP levels is quite sensitive, operating in the range of 0-50 microM glucose. In glucose-free medium, synthesis is at first suppressed but returns to a high level after 3 days as levels of the protein decrease. Synthesis and level of the Mr 69,000 HSP also were affected by glucose deprivation, but this protein appeared to be a complex of several isoelectric and Mr species, so the effects were not as dramatic. Chase experiments show that the half-lives of both the Mr 85,000 and Mr 69,000 HSPs are reduced by a factor of 2.0-2.5 after 5 days of glucose deprivation. The half-life of the Mr 69,000 HSP also was reduced by glutamine deprivation, whereas that of the Mr 85,000 HSP was essentially unaffected. This increase in turnover appears to be sufficient to account for the reduced intracellular level, thus suggesting that glucose sustains high HSP levels mainly by decreasing degradation of the proteins. Although the function of the HSPs is not known, these data support the concept that they have important roles in the general cellular economy and do not function merely as "stress" proteins.
Asparaginylglucose: Novel type of carbohydrate linkage
Authors: Wieland Felix , Heitzer Renate , Schaefer Wolfram
Published: May 2006

The
Halobacterial
cell wall glycoprotein was recently shown to contain two types of sulfated saccharides: a repetitive saccharide and a nonrepetitive saccharide composed of glucuronic acid and glucose. A new type of
N
-glycosidic linkage is found in this latter type of saccharide: glucose is
N
-glycosidically linked to the polypeptide chain through the amido nitrogen of an asparagine residue, as shown by chemical analyses, proton magnetic resonance spectroscopy, and mass spectroscopy of an isolated asparaginyl saccharide. The only
N
-glycosidic linkage known so far is between the amido nitrogen of asparagine and
N
-acetylglucosamine.