bluuebunny/crossref_metadata_2025 huggingface dataset:
Pronounced structural similarities between the small subunit ribosomal RNA genes of wheat mitochondria and Escherichia coli.
Authors: Spencer D F , Schnare M N , Gray M W
Published: May 2006

We present here the nucleotide sequence of the small subunit (18S) rRNA gene from wheat mitochondria. Aside from five discrete variable domains, this gene and the analogous (16S) rRNA gene in Escherichia coli show essentially a one-to-one correspondence in their potential secondary structures, with regions accounting for 86% of the bacterial 16S rRNA having a strict secondary structure counterpart in the mitochondrial 18S rRNA. Primary sequence identity between the two rRNAs ranges from 73% to 85% (76% overall) within regions of conserved secondary structure. Within a smaller secondary structure core common to all small subunit rRNAs, the wheat mitochondrial sequence shares substantially more primary sequence identity with the E. coli (eubacterial) sequence (88%) than with the small subunit rRNA sequences of Halobacterium volcanii (an archaebacterium) (71%) or Xenopus laevis cytoplasm (61%). Moreover, the wheat mitochondrial sequence contains a very high proportion of certain lineage-specific residues that distinguish eubacterial/plastid 16S rRNAs from archaebacterial 16S and eukaryotic cytoplasmic 18S rRNAs. These data establish that the ancestry of the wheat mitochondrial 18S rRNA gene can be traced directly and specifically to the eubacterial primary kingdom, and the data provide compelling support for a relatively recent xenogenous (endosymbiotic) origin of plant mitochondria from eubacteria-like organisms.
Homoeosis in Drosophila: the ultrabithorax larval syndrome.
Authors: Hayes P H , Sato T , Denell R E
Published: May 2006

Recent results [Morata, G. & Kerridge, S. (1981) Nature (London) 290, 778-781] have shown that early Ultrabithorax- clones transform the posterior compartments of the adult meso- and metathoracic legs to prothorax. These transformations have not been seen in Ultrabithorax homozygous larvae, which are reported to show only transformations of the metathorax and the first abdominal segment to mesothorax [Lewis, E. B. (1978) Nature (London) 276, 565-570]. However, as the ventral surface of the larva does not exhibit sufficient markers to distinguish the posterior regions of these segments, cryptic larval transformations similar to those in the adult have been suggested (by Morata and Kerridge). We have further examined larvae of wild-type and various Ultrabithorax mutant genotypes, with particular attention to the dorsal surface. We find that Ultrabithorax homozygous larvae exhibit dorsal abnormalities consistent with transformations of the anterior metathorax and anterior first abdominal segment to mesothorax and of the posterior meso- and metathorax to prothorax as predicted by Morata and Kerridge; however, the posterior of the first abdominal segment remains untransformed. We suggest that in both larvae and adults the posterior first abdominal segment remains untransformed by Ultrabithorax mutations and that the unit of development with regard to the proximal bithorax complex consists of adjoining posterior and anterior compartments from neighboring segments rather than of segments themselves.
Accelerated age-related browning of human collagen in diabetes mellitus.
Authors: Monnier V M , Kohn R R , Cerami A
Published: May 2006

The nonenzymatic glycosylation reaction that is accelerated in diabetes is the first step of the Maillard or nonenzymatic browning reaction that occurs in stored food. The glucose-protein adduct rearranges and dehydrates to form brown and fluorescent pigments, which can act as crosslinks, resulting in decreased protein solubility and altered mechanical properties. Evidence suggesting that this process occurs in vivo has been found in lens crystallins. The observation that nonenzymatic glycosylation and insolubility increases in collagen with age and diabetes led us to investigate the possible browning of human collagen. Insoluble human dura mater collagen was digested with collagenase. Absorbance at 350 nm and fluorescence at 440 nm (excitation at 370 nm) of the solubilized material was measured. A linear increase in the amounts of yellow and fluorescent material was observed with age. Samples obtained at autopsy from three type I diabetics and a young type II diabetic showed increased fluorescence and had absorbance values that corresponded to the amount of chromophore found in nondiabetics twice their age (P less than 0.025). The collagen adducts from aged and diabetic individuals had absorption and fluorescence spectra identical to those of collagen samples that underwent nonenzymatic browning with glucose in vitro. The structure of these collagen adducts is unknown. However, their likely occurrence throughout the body could explain the correlation between arterial stiffening, decreased joint mobility, and the severity of microvascular complications in type I diabetics.
Competitive inhibition by diacylglycerol of specific phorbol ester binding.
Authors: Sharkey N A , Leach K L , Blumberg P M
Published: May 2006

Emerging evidence suggests that the phorbol ester receptor in brain may be the same as the Ca2+-phospholipid-dependent protein kinase (protein kinase C). Since protein kinase C activity is stimulated by unsaturated diacylglycerol and the phorbol esters can substitute for diacylglycerol in this stimulation, we have examined the effect of diacylglycerols on phorbol ester binding. Assays were carried out with the mouse brain cytosolic phorbol ester aporeceptor, which requires phospholipids for activity. In the presence of phosphatidylserine at 0.96 mg/ml, diolein inhibited specific binding of [3H]phorbol 12,13-dibutyrate ( [3H]PBt2) in a dose-dependent fashion to less than 10% of control levels. The inhibition curves fit the curve expected for a competitive inhibitor and yielded a Ki of 3.6 +/- 0.8 micrograms/ml (n = 5) [0.38% (wt/wt) the concentration of phosphatidylserine]. Scatchard analysis confirmed the competitive nature of the inhibition. At constant phospholipid concentrations, the Ki determined for diolein was independent of the diolein concentrations over the range of 1.5-80 micrograms/ml, suggesting that the inhibition did not arise simply by perturbation of the phospholipid bilayers. The Ki of diolein was approximately proportional to the absolute phospholipid concentration. With phosphatidylserine at 4.8 micrograms/ml, for example, the Ki was 52 ng/ml (1.1% of phosphatidylserine). In addition to diolein, the short-chain saturated diacylglycerol derivatives dicaprylin and dicaproin also inhibited [3H]PBt2 binding, whereas the long-chain saturated derivatives dipalmitin and distearin were much less active. Our results suggest (i) that diacylglycerol may act as an endogenous ligand for the phorbol ester receptor and (ii) that variation in lipid composition provides a mechanism for modulating phorbol ester receptor affinity.
Intracellular free magnesium in erythrocytes of essential hypertension: relation to blood pressure and serum divalent cations.
Authors: Resnick L M , Gupta R K , Laragh J H
Published: May 2006

Intracellular levels of free Mg2+ in human erythrocytes were determined by 31P NMR spectroscopy in 26 fasted subjects and were correlated with blood pressures and serum levels of total magnesium (bound and free Mg2+) and ionized calcium from the same subjects in a seated position. Untreated hypertensive individuals consistently demonstrated lower levels of intracellular free magnesium (192 +/- 8 microM, n = 11) than either normotensive (261 +/- 9.8 microM, n = 7, P less than 0.001 vs. untreated hypertensive subjects) or hypertensive subjects whose blood pressure had been normalized on therapy (237 +/- 7.8 microM, n = 8, P less than 0.005 vs. untreated hypertensives). For all subjects, strong relationships existed between intracellular free magnesium and diastolic blood pressure (r = -0.85, P less than 0.001) and systolic blood pressure (r = -0.71, P less than 0.001). Significant relationships also were observed between intracellular free magnesium levels and extracellular serum levels of calcium ion (r = -0.77, P less than 0.001) as well as serum concentrations of total magnesium (r = 0.62, P less than 0.001). We conclude that significant depletion of intracellular free magnesium levels are apparent in erythrocytes of subjects with essential hypertension. Furthermore, the close, inverse relationship of free magnesium levels with the height of the blood pressure suggests that abnormalities of intracellular magnesium metabolism may contribute to the pathophysiology of human essential hypertension.
On the mathematical theory of globally optimal planned economic systems
Authors: Hua Loo-Keng
Published: May 2006

Applying a theorem of non-negative matrix to an economic system, we obtain the rate of growth of the system and the ratio between the sectors of production. Accordingly, we establish a positive eigenvector method for planning economics.
Detection
in vivo
of a new gene product (gene
III
) of cauliflower mosaic virus
Authors: Xiong C. , Lebeurier G. , Hirth L.
Published: May 2006

Cauliflower mosaic virus DNA contains six major open reading frames (ORFs). As only the mRNA corresponding to the transcription of gene
VI
and its translation product have been isolated, the identification in infected plants of products corresponding to the five other putative genes remains to be established. The present paper reports the detection of an ORF III product by means of antibodies raised against an NH
2
-terminal synthetic peptide of 19 amino acids corresponding to a sequence predicted from the nucleotide sequence of ORF III. The detection of this gene product raises the question of the mechanism of its expression.
Subcellular distribution of DNA-binding and non-DNA-binding 1,25-dihydroxyvitamin D receptors in chicken intestine.
Authors: Nakada M , Simpson R U , DeLuca H F
Published: May 2006

A comparison of 1,25-dihydroxyvitamin D3 receptor concentration and equilibrium dissociation constants of whole tissue, nuclear, and cytosol extracts of vitamin D-deficient chicken intestine has been carried out. The extracts had the following order of receptor concentration: nuclei greater than whole tissue greater than cytosol. The receptors in each preparation had identical equilibrium dissociation constants (Kd) for 1,25-dihydroxyvitamin D3. However, the receptor in the cytosol fraction did not bind to DNA-cellulose, while a large fraction of the receptor population in crude nuclear extract and whole tissue extract bound to DNA-cellulose and could be eluted with 0.22 M KCl, suggesting that the cytosolic form of the receptor does not possess a DNA binding site.
(Pro)insulin associates with Golgi membranes of pancreatic B cells.
Authors: Orci L , Ravazzola M , Perrelet A
Published: May 2006

The immunocytochemical demonstration of (pro)insulin on intracellular membrane compartments of the pancreatic B cell reveals that the immunolabeling detected by the protein A/gold method is associated, at the level of the Golgi apparatus, with the inner aspect of the cisternal membranes; on the secretory granules, by contrast, insulin immunoreactive sites predominate over the granule core, and very little immunoreactivity is associated with the granule membrane. The localization of (pro)insulin immunoreactivity on Golgi membranes is compatible with the presence, at this level, of specific binding sites for (pro)insulin, which could be related to the proteolytic processing or sorting out (or both) of these peptides on their way from the rough endoplasmic reticulum to the storage secretory granules.
Differentiation of human erythroid cells is associated with increased O-glycosylation of the major sialoglycoprotein, glycophorin A.
Authors: Gahmberg C G , Ekblom M , Andersson L C
Published: May 2006

Glycophorin A, the major human erythrocyte sialoglycoprotein, is found exclusively on cells of the erythroid lineage. The amino acid sequence is known, and glycophorin A isolated from mature erythrocytes contains a single N-glycosidic and 15 O-glycosidic oligosaccharides. Monoclonal antibodies against erythrocyte glycophorin A reacted weakly with erythroid precursors while a monospecific rabbit antiserum reacted strongly with immature and mature red cells. Glycophorin A was isolated from cells representing various stages of erythropoiesis in normal bone marrow, from blood cells of neonates with erythroblastosis fetalis, and from the erythroleukemic cell lines K562 and HEL before and after induced differentiation. Analysis of the oligosaccharides showed less O-glycosylation of glycophorin A in erythroid precursors. The degree of glycosylation increased concomitantly with differentiation.
Specific binding to cultured cells of 125I-labeled type beta transforming growth factor from human platelets.
Authors: Tucker R F , Branum E L , Shipley G D , Ryan R J , Moses H L
Published: May 2006

Purified type beta transforming growth factor from human platelets (TGF beta) radioiodinated with 125I-labeled Bolton and Hunter reagent was found to bind to a variety of cultured cells of both epithelial and mesenchymal origin, including normal human fibroblasts and keratinocytes. TGF beta binding sites have also been found on three mouse embryo-derived fibroblast-like cell lines with lower levels of TGF beta binding on the chemically transformed derivatives of these cell lines. A variety of human tumor cell lines was shown to have an inverse correlation between their level of TGF beta binding and their ability to form colonies in soft agar. The mouse embryo-derived AKR-2B (clone 84A) cells reached maximal binding of 125I-labeled TGF beta after 2 hr at 22 degrees C. Scatchard analysis of the equilibrium binding of TGF beta to AKR-2B (clone 84A) cells gives a Kd of 33 pM with approximately equal to 10,500 binding sites per cell. This Kd for TGF beta binding to AKR-2B (clone 84A) cells agreed well with the ED50 of 40 pM for stimulation of colony formation of these cells by TGF beta. The TGF beta binding sites on the AKR-2B cells were shown to be specific for TGF beta with no significant competition with epidermal growth factor, fibroblast growth factor, or insulin and only a small level of competition with high concentrations of platelet-derived growth factor. Partially purified preparations with TGF beta-like activity from mouse embryos and medium conditioned by mouse embryo-derived cells competed effectively for binding to the TGF beta receptor.
The sup-7(st5) X gene of Caenorhabditis elegans encodes a tRNATrpUAG amber suppressor.
Authors: Bolten S L , Powell-Abel P , Fischhoff D A , Waterston R H
Published: May 2006

In earlier studies, we identified in Caenorhabditis elegans two informational suppressors sup-5 III and sup-7 X and recently showed that these suppressors acted via an altered tRNA to suppress translational termination at amber (UAG) stop codons. We now show that the sup-7 (st5) suppressor is a tRNATrpUAG amber suppressor. These studies utilized a radiolabeled purified tRNA fraction to identify hybridizing genomic sequences in a phage genomic library. DNA sequence analysis of the hybridizing segment of one clone showed that the probe recognized a tRNATrpUGG sequence. The sup-7 gene was shown to be one of an 11 or 12 member tRNATrp family by Southern blot analysis, taking advantage of an Xba I restriction site induced in the anticodon sequence by the mutational event to suppressor. Sequence analysis of a recombinant lambda clone containing sup-7 gene proved that sup-7(st5) is a tRNATrpUAG. This conclusive proof of the nature of sup7(st5) will permit unambiguous interpretation in genetic applications, and the availability of the cloned sequences may allow the sup-7 gene to be used to select for the reintroduction of DNA into C. elegans.
Antitermination of transcription from an Escherichia coli ribosomal RNA promoter.
Authors: Holben W E , Morgan E A
Published: May 2006

The Escherichia coli lac and ara promoters and rrnC ribosomal RNA promoter-leader region were fused to lacZYA. Transcription termination signals were introduced into the lac genes of these fusions by Tn9 and IS1 insertions. Measurement of lac enzymes from upstream and downstream of the insertions showed that termination signals resulting from these insertions are very efficient when transcription begins at lac or ara promoters but are very inefficient when transcription begins at the rrnC promoter-leader region. The rrnC promoter-leader region must, therefore, modify RNA polymerase to enable it to read through transcription termination signals.
Viral integration near c-myc in 10-20% of mcf 247-induced AKR lymphomas.
Authors: Li Y , Holland C A , Hartley J W , Hopkins N
Published: May 2006

We induced AKR thymomas by injecting retrovirus MCF 247 that we had tagged with a fragment of phage lambda in its U3 region. About 10-20% of the 26 primary tumors studied showed both rearranged and germline c-myc bands in Southern blots. The rearranged c-myc genes were cloned from two of the tumors and studied by Southern analysis in two others. In all four cases, rearrangement was due to integration of MCF 247 proviruses about 2 kilobases (kb) 5' of the three c-myc exons and in the opposite transcriptional orientation. Thus, viral integrations were clustered within a 1-kb range in the tumors that revealed c-myc rearrangements. Three of the four proviruses appeared to be intact, whereas the fourth had an internal deletion of about 3 kb.
Selective association of N-methyl aspartate and quisqualate types of L-glutamate receptor with brain postsynaptic densities.
Authors: Fagg G E , Matus A
Published: May 2006

Recognition sites for the excitatory neurotransmitter, L-glutamate, were studied in synaptic plasma membranes and postsynaptic densities (PSDs) isolated from rat brains. The results demonstrate (i) that L-glutamate binding sites may be resolved into three distinct subtypes (categories A1, A2, and A4), each corresponding to an electrophysiologically identified receptor class, and (ii) that the N-methyl aspartate (A1) and quisqualate (A2) receptor types are selectively associated with PSDs. L-[3H]Glutamate bound to an apparently homogeneous population of sites in PSDs with a Kd of 3.39 X 10(-7) M and a Bmax (maximum number of binding sites) of 6.1 pmol/mg of protein. Inhibition studies demonstrated that these sites could be resolved into two distinct subtypes. N-Methyl aspartate maximally inhibited 58% of PSD-located L-glutamate binding sites with a Ki of 7.2 X 10(-6) M (the A1 site), and quisqualate inhibited 42% with a Ki of 1.1 X 10(-6) M (the A2 site); the effects of both substances were additive. Experiments with a range of acidic amino acid analogues indicated that the ligand selectivities of these two binding sites conformed to those of the N-methyl D-aspartate and quisqualate receptor classes defined electrophysiologically. The Cl--dependent population of L-glutamate binding sites (the A4 site), which predominates in synaptic membranes, was absent from isolated PSDs.
Coagulation factors V and VIII and ceruloplasmin constitute a family of structurally related proteins.
Authors: Church W R , Jernigan R L , Toole J , Hewick R M , Knopf J , Knutson G J , Nesheim M E , Mann K G , Fass D N
Published: May 2006

Computer searches of the National Biomedical Research Foundation protein and nucleic acid sequence data bases using the NH2 terminus of the bovine factor Va 94-kilodalton heavy chain, the NH2 terminus of the 74-kilodalton factor Va light chain, and an internal 98-residue segment of porcine factor VIII revealed that both bovine factor V and porcine factor VIII are statistically homologous to human ceruloplasmin. The NH2-terminal segment of bovine factor Va heavy chain is homologous to three segments of ceruloplasmin sequence starting at residues 1, 351, and 713; the NH2-terminal sequence of bovine factor Va light chain is homologous to the same human ceruloplasmin sequence segments beginning at residues 1, 349, and 711. The longer porcine factor VIII sequence is homologous to three segments of human ceruloplasmin, residues 1-77, 400-433, and 683-791. These data indicate that factor V, factor VIII, and ceruloplasmin comprise a group of evolutionarily linked protein structures that possibly resulted from multiplication of ancestral precursor genes.
Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine.
Authors: Santi D V , Norment A , Garrett C E
Published: May 2006

DNA containing 5-azacytosine (azaC) has previously been shown to be a potent inhibitor of DNA-cytosine methyltransferases. In this report, we describe experiments which demonstrate that azaC-DNA forms a covalent complex with Hpa II methylase, a bacterial enzyme that methylates the internal C of C-C-G-G sequences. The complex does not undergo detectable dissociation over at least 3 days and is stable to denaturation with NaDodSO4. After extensive digestion of the complex with DNase and phosphodiesterase, gel filtration gave the methylase bound to approximately one equivalent of azaC; the digested complex had an apparent molecular weight similar to that of the native enzyme. Although prior treatment of azaC-DNA with Hpa II endonuclease had only a slight effect on binding of the methylase, treatment with Msp I endonuclease, which also cleaves at C-C-G-G sequences, resulted in a significant reduction in binding; this indicates that azaC residues in the recognition sequence of Hpa II are an important component in the covalent interaction of the methylase. However, since there was residual binding it is possible that azaC residues elsewhere in DNA also covalently bind to the methylase. These results provide an explanation of why azaC-DNA is such a potent inhibitor of cytosine methyltransferases and how the incorporation of such low levels of azaC into DNA can result in dramatic decreases in the methylation of cytosine. Finally, consideration of the probable catalytic mechanism of cytosine methylases and the chemical properties of azaC suggests that the inhibition is, at least in part, an active-site directed process and permits a proposal for the structure of the covalent complex.
Characterization of a somatostatin-28 containing the (Tyr-7, Gly-10) derivative of somatostatin-14: a terminal active product of prosomatostatin II processing in anglerfish pancreatic islets.
Authors: Morel A , Gluschankof P , Gomez S , Fafeur V , Cohen P
Published: May 2006

Anglerfish (Lophius piscatorius) Brockmann organs contain a form of somatostatin-14, identical to the hypothalamic tetradecapeptide, and two distinct forms of somatostatin-28, which can be separated by reversed-phase high-pressure liquid chromatography (HPLC). Analysis of the NH2-terminal amino acid sequence and comparison of the ability to incorporate 125I indicate that one of these forms corresponds to an octacosapeptide including in its sequence the (Tyr-7, Gly-10) derivative of somatostatin-14 (somatostatin II). Exposure of this somatostatin-28 species to an endopeptidase activity from the rat brain cortex generates a peptide immunologically related to somatostatin and undistinguishable from synthetic (Tyr-7, Gly-10) somatostatin-14 II by HPLC. This somatostatin-28 II exhibits a potent inhibitory effect on growth hormone release by rat anterior pituitary cells, comparable to the other somatostatin-28 form. Since (Tyr-7, Gly-10) somatostatin-14 II cannot be detected in anglerfish pancreatic islets, these results indicate that somatostatin-28 II represents the terminal active product of prosomatostatin II processing, whose structure was predicted from the cDNA nucleotide sequence corresponding to the second mRNA cloned from anglerfish Brockmann organs [Hobart, P., Crawford, R., Shen, L. P., Pictet, R. & Rutter, W. J. (1980) Nature (London) 288, 137-141].
Extreme instability of myc mRNA in normal and transformed human cells.
Authors: Dani C , Blanchard J M , Piechaczyk M , El Sabouty S , Marty L , Jeanteur P
Published: May 2006

To address the possibility that the expression of the myc gene might be regulated at a post-transcriptional level, we have investigated the half-life of myc mRNA in various cells. Our survey included normal human embryonic fibroblasts as well as transformed human cells of various origins: cervix carcinoma (HeLa), breast carcinoma (MCF7), Burkitt lymphoma (Daudi), and promyelocytic leukemia (HL60). All these cells revealed an extreme instability of myc mRNA (half-life, approximately equal to 10 min), suggesting that the control of myc mRNA degradation might be a general means (although not necessarily exclusive) of regulating both the level and the timing of myc gene expression. Inhibition of protein synthesis resulted in a dramatic stabilization of myc mRNA in HeLa, MCF7, and HL60 cells, suggesting that the controlling element might itself be, at least in these cells, a protein of rapid turnover. This finding opens the way to studying the mechanism of myc mRNA inactivation in these different cell types. However, protein synthesis inhibition had no effect on myc mRNA instability in other transformed (Daudi) cell lines as well as normal embryonic human fibroblasts. These different types of behavior suggest that the post-transcriptional control of myc gene expression might involve multiple factors that would be differently affected in various cell types.
Plasmid transfer from Escherichia coli to Bacteroides fragilis: differential expression of antibiotic resistance phenotypes.
Authors: Guiney D G , Hasegawa P , Davis C E
Published: May 2006

A unique shuttle plasmid, pDP1, has been constructed to mediate gene transfer between Escherichia coli and the Gram-negative anaerobe Bacteroides fragilis. pDP1 contains the pBR322 replicon and the Bacteroides clindamycin resistance plasmid pCP1 linked to the transfer origin of the broad host range plasmid RK2. pDP1 can be transferred from E. coli to B. fragilis by the RK2 conjugation system even though RK2 itself is not maintained in the Bacteroides recipients. The antibiotic resistance and replication functions of pDP1 have been mapped by deletion analysis, and a 5-kilobase portion of the plasmid has been identified as the essential region for maintenance in Bacteroides. Comparison of the resistance conferred by pDP1 on E. coli and B. fragilis shows that antibiotic resistance genes are expressed differently in aerobic and anaerobic bacteria. These results document the feasibility of gene transfer from E. coli to B. fragilis and demonstrate the usefulness of this conjugation system to study genetic structure and expression in Bacteroides.
On applications of diophantine approximations
Authors: Chudnovsky G. V.
Published: May 2006

This paper is devoted to the study of the arithmetic properties of values of
G
-functions introduced by Siegel [Siegel, C. L. (1929)
Abh. Preuss. Akad. Wiss. Phys.-Math. Kl.
1]. One of the main results is a theorem on the linear independence of values of
G
-functions at rational points close to the origin. In this theorem, no conditions are imposed on the
p
-adic convergence of a
G
-function at a generic point. The theorem finally realizes Siegel's program on
G
-function values outlined in his paper.
Salt-induced Z-A-Z transition sequence in the mixed ribo-deoxyribo copolymer poly(rG-dC) X poly(rG-dC).
Authors: Wu H Y , Behe M J
Published: May 2006

The left-handed helical conformation of various polynucleotides has been seen in solution at higher salt concentration than has the right-handed helical conformation of the corresponding polymer. We report here, however, studies of conformational transitions in the mixed polymer poly-(rG-dC) X poly(rG-dC). This polynucleotide appears to exist in a Z conformation under low-salt conditions, converts to an A helix at intermediate salt, and reverts to a left-handed helix at high-salt concentration.
Changing patterns of gene expression during sporulation in yeast.
Authors: Kurtz S , Lindquist S
Published: May 2006

Analysis of RNAs isolated from the yeast Saccharomyces cerevisiae reveals a dramatic series of changes in protein coding sequences during sporulation. Shortly after transfer to sporulation medium, mRNAs for certain proteins are repressed while a broad array of mRNAs for other proteins is induced. Superimposed on this general increase in transcriptional activity is the very strong induction of a particular subset of heat shock mRNAs, the same subset that is induced during the normal course of oogenesis in Drosophila. At distinct times later in sporulation, two sets of abundant mRNAs are coordinately induced. Unlike the earlier changes in the message complement, these changes are unique to sporulating cells. As asci mature, one set of sporulation-specific RNAs is selectively degraded. The second set, as well as the broad array of mRNAs induced earlier in development, is retained in a highly stable and fully translatable form.
Na+-H+ exchange in gastric glands as measured with a cytoplasmic-trapped, fluorescent pH indicator.
Authors: Paradiso A M , Tsien R Y , Machen T E
Published: May 2006

We have used the pH-sensitive, fluorescent, cytoplasmic-trapped dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to identify Na+-H+ exchange in gastric glands isolated from rabbit stomachs by high-pressure perfusion and collagenase digestion. The fluorescence of BCECF-loaded glands was calibrated in terms of cytosolic pH (pHc) by permeabilizing the cell membranes and titrating the extracellular solution to different pH values. In one set of experiments in Cl--free solutions, glands were treated with 0.1 mM ouabain for 45 min to increase cellular cytosolic molar sodium ion concentration [( Na+]c) to high levels. Subsequent suspension of these cells in a Na+-free Ringer's solution (to generate [Na+]c greater than [Na+]o) caused cells to acidify rapidly (t1/2 approximately equal to 60 sec) from pHc approximately equal to 7.15 to pHc approximately equal to 6.55. Subsequent addition of 100 mM Na+ or Li+, but not K+, caused cells rapidly to increase pHc (t1/2 approximately equal to 30 sec) toward the control value. These changes of pHc were blocked when ouabain-treated glands had been preequilibrated for 10 min with 1 mM amiloride, and this block was overcome by adding 10 microM monensin (an ionophore that artificially exchanges Na+ for H+). In another set of experiments in Cl--containing Ringer's solution, glands were acid-loaded by treatment with 30 mM NH4Cl for 4 min, followed by washing the NH4Cl from the solutions. Under these conditions, pHc decreased from 7.02 to approximately equal to 6.5; subsequent alkalinization of cells back to control pHc was stimulated by Na+ (t1/2 approximately equal to 60 sec), but not K+, and was inhibited by 1 mM amiloride. This amiloride block also was overcome by further addition of 10 microM monensin. We conclude that gastric glands contain a Na+-H+ exchanger that appears independent of Cl-, not activated by K+, and blocked by 1 mM amiloride. This exchanger is likely localized to the serosal membrane of gland cells. Na+-H+ exchange may play an important role in regulation of pHc in oxyntic and chief cells exposed to high luminal acidity, where back diffusion of H+ into cells may occur at rapid rates.
Direct transfection of viral and plasmid DNA into the liver or spleen of mice.
Authors: Dubensky T W , Campbell B A , Villarreal L P
Published: May 2006

A method for the direct transfection of polyoma viral DNA and polyoma-plasmid recombinant DNA into the liver or spleen of newborn or adult mice was developed. Calcium phosphate-precipitated DNA was injected directly into mouse organs in combination with hyaluronidase and collagenase. Transfected DNA was shown to replicate at moderate efficiency, relative to direct infection of organs with virus. Transfection with viral DNA rapidly led to an acute infection. A polyoma-bacterial plasmid recombinant DNA also was shown to replicate when transfected into mice. With this plasmid, however, genomic-length polyoma DNA rapidly recombined away from the bacterial component and replicated as viral DNA. This method should allow the direct determination of the biological activity of a cloned DNA within a mouse organ.
Role of a disulfide bridge in the immune function of major histocompatibility class I antigen as studied by in vitro mutagenesis.
Authors: Shiroishi T , Evans G A , Appella E , Ozato K
Published: May 2006

Polymorphic major histocompatibility class I antigens have highly conserved disulfide bridges in the second and third external domains. To study the role of a disulfide bridge, we have introduced a mutation into the mouse H-2Ld gene by oligonucleotide-directed site-specific mutagenesis, disrupting the disulfide bridge in the second domain of the protein by changing cysteine at amino acid position 101 into serine. Upon introduction of the mutant gene into L cells, the mutant transplantation antigens were synthesized, inserted into the membrane, and displayed on the cell surface, indicating that the disulfide bridge is not essential for surface expression of the H-2 antigen. Binding studies carried out with 16 monoclonal antibodies specific for the H-2Ld antigen showed that most of the allodeterminants are lost or greatly altered in the mutant antigen. Further, almost complete loss of the recognition by H-2Ld-specific alloreactive cytotoxic T cells was observed. These results indicate that polymorphic determinants are dependent on a protein folding pattern dictated by the disulfide bridge. However, two antibodies previously found to react with antigenic sites present in the first and third domains were reactive with the mutant, implying an element of domain independence with respect to the determinants recognized by these antibodies.
Antigen presentation by murine and human cells to a murine T-cell hybridoma: demonstration of a restriction element associated with a major histocompatibility complex class II determinant(s) shared by both species.
Authors: Waters S J , Winchester R J , Nagase F , Thorbecke G J , Bona C A
Published: May 2006

A CB6F1 murine T-cell hybridoma, FN1-18, secreting interleukin 2 in response to presentation of keyhole limpet hemocyanin (KLH) by syngeneic cells in the context of an I-E gene complementation product, also exhibited a major histocompatibility complex-restricted response to KLH presented by human leukocytes. The murine restriction element was I-E beta b or k E alpha d or k. The KLH-induced interleukin 2 production was inhibited by the monoclonal antibody 17-3-3s with similar but not identical anti-I-E specificity. This antibody reacted with human Ia-like antigens on T-depleted mononuclear cells in 10 of 30 humans, as assessed by immunofluorescence. The presence of the epitope recognized by antibody 17-3-3s was closely correlated with MT3 allospecificity. The ability of human cells to present KLH to the murine hybridoma strongly correlated (r = 0.768) with the expression of this epitope and was also inhibited by 17-3-3s. Another monoclonal antibody, 109d6, of the same isotype and with related but not identical MT3 specificity, caused only limited inhibition. The ability of human cells to present KLH segregated as a dominant trait linked to the major histocompatibility complex. The results indicate that the restriction element recognized by FN1-18 on both human and mouse cells is a determinant closely related to but not identical with the antigenic determinant to which 17-3-3s binds. This determinant is not influenced by the considerable species differences in the remainder of the Ia molecules.
Specific binding of erythropoietin to spleen cells infected with the anemia strain of Friend virus.
Authors: Krantz S B , Goldwasser E
Published: May 2006

Tritiated erythropoietin with full biological activity has been prepared, and a relatively homogeneous population of enriched progenitor cells that respond to the hormone has been generated by infection of mice with the Friend virus that produces anemia. These cells, obtained from the spleens of infected mice, develop into mature erythroblasts and erythrocytes in the presence of erythropoietin. We have measured the binding of erythropoietin to these target cells; 62% of the binding was inhibited by excess unlabeled erythropoietin, but no inhibition occurred with albumin, serum, or a variety of growth factors and glycoproteins. Apparent equilibrium was reached by 2 hr at 37 degrees C and by 3.5-4 hr at 10 degrees C. The extent of specific binding increased linearly with cell concentration. In binding experiments at 10 degrees C, apparent saturation of specific binding occurred at approximately equal to 8.7 nM. Scatchard analysis showed a single class of binding sites. The dissociation constant is 5.2 nM with an average of 660 binding sites per cell. At 0.06 nM, where most of the cells are induced to terminally differentiate in vitro, an average of only 8 erythropoietin molecules bound per cell. These studies indicate that erythropoietin attaches to specific binding sites, which are most likely receptors since they manifest high affinity and specificity, and that the biologic effect of the hormone may be produced by attachment of a very small number of erythropoietin molecules.
Lipoprotein lipase is produced, regulated, and functional in rat brain.
Authors: Eckel R H , Robbins R J
Published: May 2006

Lipoprotein lipase (LP lipase, triacylglycero-protein acylhydrolase EC 3.1.1.34) activity was found in four dissimilar brain regions (hypothalamus, cortex, cerebellum, and midbrain) of adult male rats. Progressive accumulation of LP lipase activity in cultured fetal rat hypothalamic cells was also observed, indicating de novo synthesis of the lipase. The brain LP lipase activity was serum-dependent and was inhibited by 1 M NaCl and by protamine sulfate. Kinetic analysis revealed an apparent Km of 0.79 mM very similar to that of rat adipose tissue LP lipase. That the lipase was functioning in the cultured brain cells was indicated by uptake and incorporation of radioactivity from tri[( 1-14C]oleoyl)glycerol into cellular triacylglycerols, and into more polar lipids, such as phosphatidylcholine. Furthermore, brain LP lipase activity in adult rats was decreased in all four regions examined, most significantly in the hypothalamus, after 72 hr of food deprivation. Thus, authentic LP lipase is present in adult rat brain and can be synthesized by isolated brain cells in vitro. LP lipase also mediates the uptake of triacylglycerol fatty acids and their subsequent incorporation into cellular lipids of cultured brain cells. Decreased brain LP lipase activity after fasting suggests that this enzyme may be regulated by metabolic or nutritional factors. Because the largest changes in LP lipase activity in response to food deprivation occurred in the hypothalamus, the enzyme may have a role in hypothalamic control of food intake or in body-weight regulation.
Carotenoids and retinol: their possible importance in determining longevity of primate species.
Authors: Cutler R G
Published: May 2006

Aging and cancer share a number of characteristics. This has led to the hypothesis that species' differences in longevity may be governed in part by the same mechanisms as those processes governing species' differences in their age-dependent probability of developing cancer. Much evidence has indicated that beta-carotene and retinol may be important natural anticarcinogens. Accordingly, they also may be important antiaging agents. This possibility has been tested by determining if a positive correlation exists between the concentration of carotenoids and retinol in serum and brain tissue with the maximal life-span potential of mammalian species. The results show a significant positive correlation for the carotenoids but not for retinol. These results suggest that the carotenoids may be biologically active as protective agents against cancer and as longevity determinants. Retinol appears to be less important in these functions.
Protein export in Escherichia coli requires a soluble activity.
Authors: Müller M , Blobel G
Published: May 2006

By using a reconstituted cell-free system we have demonstrated the existence of a soluble activity that is required for the export of proteins in Escherichia coli. This export factor sediments at about 12 S. It has been partially purified by passage through an omega-NH2-butylagarose column and by salt elution off a DEAE matrix. The export factor does not contain 6S RNA.
Leukemia-derived growth factor (non-interleukin-2) produced by murine lymphoma T-cell lines.
Authors: Hays E F , Goodrum D , Bessho M , Kitada S , Uittenbogaart C H
Published: May 2006

Autocrine growth factor activity was found in supernatants of AKR T-cell lymphoma lines cultured in serum-free medium. This factor was designated leukemia-derived growth factor (LDGF). Active supernatants stimulated the growth of the AKR murine T-cell lymphoma line SL 12, its cloned derivatives, and all other murine T-cell lymphoma lines tested. Growth factor activity in conditioned medium was found to be different from interleukin 2 (IL-2) and several other known growth factors. LDGF was able to stimulate growth of the human leukemia T-cells MOLT4f, and the LDGF from MOLT4f cells stimulated the mouse cells. Because mouse T-cell lymphoma lines produced and respond to this factor, it may support the continued proliferation of these cells and could be responsible for their malignant in vivo properties.
Monoclonal derivation of mouse myeloid and lymphoid lineages from totipotent hematopoietic stem cells experimentally engrafted in fetal hosts.
Authors: Mintz B , Anthony K , Litwin S
Published: May 2006

Mutant mouse fetuses with a hematopoietic stem cell defect were injected with a mixture of two normal strains of fetal liver cells to test the possibility of seeding with single stem cells and of deriving all hematopoietic lineages clonally. Recipients were either Wf/Wf, with a mild endogenous defect offering only marginal selective advantage to a normal donor cell, or W/W, with a severe defect. Among 11 Wf/Wf animals with long-term grafts, 8 had only one or the other of the donor strains. Some of these individuals must have been seeded by only a single donor cell (P = 0.1); the frequency of this event was at least 20% (90% confidence) and most likely 50% of the cases. Cell-specific strain markers in myeloid and lymphoid lineages reinforced the likelihood that renewal and differentiation had occurred from a totipotent hematopoietic stem cell. In a smaller W/W group, some hosts were seeded by at most two cells (P = 0.1), and single-cell seeding could not be ruled out. The experiment allows stem cell pedigrees to be examined during the normal developmental progression. In both groups observed here, some mice displayed a regular and complementary rise and fall in proportions of cells of different genotypes, thereby suggesting clonal succession in a hierarchy of stem cell compartments. This transplant system also offers advantages for future experiments on regulated expression in vivo of genes transferred (in vitro) into totipotent hematopoietic stem cells.
Genetic properties and chromatin structure of the yeast gal regulatory element: an enhancer-like sequence.
Authors: Struhl K
Published: May 2006

DNA molecules created by fusing a 365-base-pair segment of yeast DNA encoding the galactose-regulated upstream promoter element (gal) to a set of derivatives that systematically delete sequences upstream from the his3 gene are introduced in single copy back into the yeast genome precisely at the his3 locus and then assayed for transcription. Fusions of the gal regulatory element to his3 derivatives containing all normal mRNA coding sequences but lacking essentially the entire promoter region fail to express his3 under any growth conditions. Fusions to derivatives lacking the his3 upstream promoter element but containing the "TATA box" place his3 expression under gal control--i.e., extremely high RNA levels in galactose-containing medium and essentially no his3 RNA in glucose-containing medium. However, of the two normal his3 initiation sites, only the downstream one is activated by the gal element. In fusions of this type, neither the orientation of the gal element nor the distance between the element and the his3 TATA box affects the level or the initiation points of transcription. However, the gal element does not influence transcription when placed 100 or 300 base pairs downstream from the normal mRNA start sites. Fusions to derivatives containing the entire his3 promoter region restore the basal level of his3 transcription in glucose-grown cells, and both transcriptional initiation sites are used. Furthermore, RNA levels in galactose-grown cells, although somewhat higher than in glucose-grown cells, are significantly below the fully induced level. The distance from his3 coding sequences does not affect RNA levels, suggesting that specific sequences, possibly corresponding to the his3 upstream promoter element, reduce the ability of the gal element to activate transcription. Analysis of chromatin from some of these strains indicates a DNase I-hypersensitive site(s) in the middle of the gal element. However, this structural feature is not correlated with transcriptional initiation because it is found when cells are grown in glucose medium and also in derivatives lacking a TATA box. Thus, the gal upstream element possesses most, but not all, of the properties of viral and cellular enhancer sequences of higher eukaryotes. In addition, it appears that the his3 and gal upstream sequences represent two distinct classes of promoter elements, which activate transcription from different initiation sites.
Nucleotide sequence of human monocyte interleukin 1 precursor cDNA.
Authors: Auron P E , Webb A C , Rosenwasser L J , Mucci S F , Rich A , Wolff S M , Dinarello C A
Published: May 2006

Interleukin 1 (IL-1) is a protein with several biological activities regulating host defense and immune responses. We report here the isolation of human IL-1 cDNA. It encodes a precursor polypeptide of 269 amino acids (30,747 Mr). mRNA isolated by hybridization to this cDNA was translated in a reticulocyte cell-free system, yielding immunoprecipitable IL-1. Furthermore, this hybrid-selected mRNA was injected into Xenopus laevis oocytes, which subsequently secreted biologically active IL-1. The cDNA nucleotide sequence suggests that IL-1 is initially translated as a precursor molecule that is subsequently processed into the 15,000-20,000 Mr protein usually associated with IL-1 activity.
Human interleukin 2 can promote the growth and differentiation of single hapten-specific B cells in the presence of specific antigen.
Authors: Pike B L , Raubitschek A , Nossal G J
Published: May 2006

B lymphocytes specifically reactive to the hapten fluorescein (FLU) were prepared from normal adult murine spleen by the hapten-gelatin affinity procedure. They were placed in 10 microliter of microcultures singly or in small numbers in the absence of any feeder, filler, or accessory cell. The "T cell-independent" antigen FLU-conjugated polymerized flagellin (FLU-POL) or a selected batch of FLU-conjugated Ficoll were used, and these stimulated division and differentiation only in the concomitant presence of lymphokines acting as B-cell growth and differentiation factors (BGDF). It was found that human interleukin 2 (IL-2), prepared by recombinant DNA technology (r-IL-2), was an effective, albeit rather weak, BGDF in this system. When an IL-2-free source of BGDF was used with the antigenic stimulus, addition of r-IL-2 did not augment the response, nor did removal of IL-2 from the crude lymphokine mixture diminish the BGDF activity.