bluuebunny/crossref_metadata_2025 huggingface dataset:
Adjustments of photosystem stoichiometry in chloroplasts improve the quantum efficiency of photosynthesis.
Authors: Chow W S , Melis A , Anderson J M
Published: May 2006

The efficiency of photosynthetic electron transport depends on the coordinated interaction of photosystem II (PSII) and photosystem I (PSI) in the electron-transport chain. Each photosystem contains distinct pigment-protein complexes that harvest light from different regions of the visible spectrum. The light energy is utilized in an endergonic electron-transport reaction at each photosystem. Recent evidence has shown a large variability in the PSII/PSI stoichiometry in plants grown under different environmental irradiance conditions. Results in this work are consistent with the notion of a dynamic, rather than static, thylakoid membrane in which the stoichiometry of the two photosystems is adjusted and optimized in response to different light quality conditions. Direct evidence is provided that photosystem stoichiometry adjustments in chloroplasts are a compensation strategy designed to correct unbalanced absorption of light by the two photosystems. Such adjustments allow the plant to maintain a high quantum efficiency of photosynthesis under diverse light quality conditions and constitute acclimation that confers to plants a significant evolutionary advantage over that of a fixed photosystem stoichiometry in thylakoid membranes.
Uric acid is a major antioxidant in human nasal airway secretions.
Authors: Peden D B , Hohman R , Brown M E , Mason R T , Berkebile C , Fales H M , Kaliner M A
Published: May 2006

Airway mucosal surfaces are potentially subjected to a variety of oxidant stresses. Airway submucosal glands secrete a variety of compounds that may protect the airways from injury. Cholinergically induced nasal submucosal gland secretion has recently been found to contain a low molecular weight nasal antioxidant. In this report, the isolation and identification of this nasal secretory antioxidant are described. Concentrated, cholinergically induced human nasal secretions were fractionated through a 10-kDa sieve and subjected to DEAE anion-exchange chromatography. Fractions containing antioxidant activity were subjected to gel filtration with Bio-Gel P-2 gel (resolution range, 200-2000 Da). The resultant antioxidant fractions were then desalted by gel filtration over the same column equilibrated in HPLC-grade water, yielding only a single peak with antioxidant activity. The absorption spectrum of the purified antioxidant revealed peaks at 238 and 292 nm at pH 7. These peaks shifted to 230 and 280 nm in 0.1 M HCl and 226 and 296 nm in 0.1 M NaOH. Sodium borohydride reduction of the antioxidant had no effect on the UV absorption, whereas platinum-catalyzed hydrogenation ablated all absorption peaks. Uric acid had identical absorption peaks and showed the same chromatographic behavior as the nasal antioxidant activity on both gel filtration and DEAE columns. Uricase (which degrades uric acid) metabolized both uric acid and the purified antioxidant. Uric acid was shown to have antioxidant activity at concentrations greater than 1.5 microM. These data indicate that nasal secretions contain uric acid that serves as an antioxidant.
Transforming growth factor beta stimulates mammary adenocarcinoma cell invasion and metastatic potential.
Authors: Welch D R , Fabra A , Nakajima M
Published: May 2006

The experimental metastatic potential of 13762NF mammary adenocarcinoma clone MTLn3 was tested after pretreatment in serum-free medium containing transforming growth factor (TGF) beta 1 at 0-5000 pg/ml. Lung colonies were measured 2 weeks after inoculation in syngeneic F344 rats, and a bell-shaped dose-response curve with 2- to 3-fold increase in number of surface lung metastases was seen. Maximal enhancement occurred at the 50 pg/ml dose level. The effect was specific because addition of neutralizing anti-TGF-beta antibody blocked the stimulatory activity at all levels of TGF-beta 1 pretreatment, but when antibody was given alone, neutralizing anti-TGF-beta antibody had no effect on untreated cells. Increased metastatic potential appears to be from an increased propensity of cells to extravasate as tested in the membrane invasion culture system. MTLn3 cells penetrated reconstituted basement-membrane barriers 2- to 3.5-fold more than did untreated control cells, depending upon length of TGF-beta 1 exposure. Increased invasive potential is apparently due, in part, to a 2- to 6-fold increase in type IV collagenolytic (gelatinolytic) and a 2.4-fold increase in heparanase activity. TGF-beta 1 treatment of MTLn3 cells did not alter their growth rate or morphology in the presence of serum; however, growth was inhibited in serum-free medium. Likewise, adhesion to human umbilical vein endothelial cell monolayers or to immobilized reconstituted basement membrane or fibronectin matrices was unchanged. These results suggest that TGF-beta 1 may modulate metastatic potential of mammary tumor cells by controlling their ability to break down and penetrate basement-membrane barriers.
Interplant communication: airborne methyl jasmonate induces synthesis of proteinase inhibitors in plant leaves.
Authors: Farmer E E , Ryan C A
Published: May 2006

Inducible defensive responses in plants are known to be activated locally and systemically by signaling molecules that are produced at sites of pathogen or insect attacks, but only one chemical signal, ethylene, is known to travel through the atmosphere to activate plant defensive genes. Methyl jasmonate, a common plant secondary compound, when applied to surfaces of tomato plants, induces the synthesis of defensive proteinase inhibitor proteins in the treated plants and in nearby plants as well. The presence of methyl jasmonate in the atmosphere of chambers containing plants from three species of two families, Solanaceae and Fabaceae, results in the accumulation of proteinase inhibitors in leaves of all three species. When sagebrush, Artemisia tridentata, a plant shown to possess methyl jasmonate in leaf surface structures, is incubated in chambers with tomato plants, proteinase inhibitor accumulation is induced in the tomato leaves, demonstrating that interplant communication can occur from leaves of one species of plant to leaves of another species to activate the expression of defensive genes.
Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme.
Authors: Bredt D S , Snyder S H
Published: May 2006

Nitric oxide mediates vascular relaxing effects of endothelial cells, cytotoxic actions of macrophages and neutrophils, and influences of excitatory amino acids on cerebellar cyclic GMP. Its enzymatic formation from arginine by a soluble enzyme associated with stoichiometric production of citrulline requires NADPH and Ca2+. We show that nitric oxide synthetase activity requires calmodulin. Utilizing a 2',5'-ADP affinity column eluted with NADPH, we have purified nitric oxide synthetase 6000-fold to homogeneity from rat cerebellum. The purified enzyme migrates as a single 150-kDa band on SDS/PAGE, and the native enzyme appears to be a monomer.
Murine beta 1,4-galactosyltransferase: both the amounts and structure of the mRNA are regulated during spermatogenesis.
Authors: Shaper N L , Wright W W , Shaper J H
Published: May 2006

Previously we have shown that the gene encoding murine beta 1,4-galactosyltransferase (beta 1,4-GT; UDPgalactose:N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D-galactosyltransferase, EC 2.4.1.38) is unusual in that it specifies two sets of mRNAs of about 3.9 and 4.1 kilobases (kb). Translation of the 3.9- and 4.1-kb mRNAs results in the predicted synthesis of two related membrane-bound forms of the protein of 386 amino acids (short form) and 399 amino acids (long form), respectively. In this study we have examined the expression of beta 1,4-GT during murine spermatogenesis. Spermatogonia contain a 4.1-kb transcript that is comparable in size to the beta 1,4-GT mRNA identified in somatic cells. During differentiation from spermatogonia (2n) to pachytene spermatocytes (4n), the amount of beta 1,4-GT mRNA is reduced to barely detectable levels. Continued differentiation to round spermatids (n) is coincident with a renewed production of beta 1,4-GT mRNA to levels comparable with those detected in spermatogonia. However, the characteristic 4.1-kb mRNA detected in spermatogonia is replaced by two truncated transcripts of 2.9 and 3.1 kb. By S1 nuclease analysis, the 2.9- and 3.1-kb transcripts were shown to encode the same open reading frame as the 4.1-kb transcript found in somatic cells. The shorter round spermatid transcripts arise as a consequence of the use of alternative poly(A) signals. Lastly, we show that, in direct contrast to all somatic tissues and cell lines examined to date, male germ cells synthesize only the long form of the beta 1,4-GT polypeptide.
Juvenile hormone receptors in insect larval epidermis: identification by photoaffinity labeling.
Authors: Palli S R , Osir E O , Eng W , Boehm M F , Edwards M , Kulcsar P , Ujvary I , Hiruma K , Prestwich G D , Riddiford L M
Published: May 2006

Tritiated photoaffinity analogs of the natural lepidopteran juvenile hormones, JH I and II [epoxy[3H]bishomofarnesyl diazoacetate ([3H]EBDA) and epoxy[3H]homofarnesyl diazoacetate ([3H]EHDA)], and of the JH analog methoprene [[3H]methoprene diazoketone ([3H]MDK)] were synthesized and used to identify specific JH binding proteins in the larval epidermis of the tobacco hornworm (Manduca sexta). EBDA and EHDA specifically photolabeled a 29-kDa nuclear protein (pI 5.8). This protein and a second 29-kDa protein (pI 6.0) were labeled by MDK, but excess unlabeled methoprene or MDK only prevented binding to the latter. These 29-kDa proteins are also present in larval fat body but not in epidermis from either wandering stage or allatectomized larvae, which lack high-affinity JH binding sites. A 29-kDa nuclear protein with the same developmental specificity as this JH binder bound the DNA of two larval endocuticle genes. A 38-kDa cytosolic protein was also specifically photolabeled by these photoaffinity analogs. The 29-kDa nuclear protein is likely the high-affinity receptor for JH that mediates its genomic action, whereas the 38-kDa cytosolic protein may serve as an intracellular carrier for these highly lipophilic hormones and hormone analogs.
Possible deletion of a developmentally regulated heavy-chain variable region gene in autoimmune diseases.
Authors: Yang P M , Olsen N J , Siminovitch K A , Olee T , Kozin F , Carson D A , Chen P P
Published: May 2006

Several autoantibody-associated variable region (V) genes are preferentially expressed during early ontogenic development, suggesting strongly that they are of developmental and physiological importance. As such, it is possible that polymorphisms in one or more of these genes may alter susceptibility to autoimmune disease. We have searched extensively for a probe related to a developmentally regulated V gene that has the power to differentiate among highly homologous V genes in human populations. Using such a probe (i.e., Humhv3005/P1) related to both anti-DNA and anti-IgG autoantibodies, we studied restriction fragment length polymorphisms in patients with rheumatoid arthritis and systemic lupus erythematosus and found an apparent heavy-chain V (VH) gene deletion that was nearly restricted to the autoimmune patients. These data suggest that deletions of physiologically important VH genes may increase the risk of autoimmunity through indirect effects on the development and homeostasis of the B-cell repertoire.
Steroids induce acetylcholine receptors on cultured human muscle: implications for myasthenia gravis.
Authors: Kaplan I , Blakely B T , Pavlath G K , Travis M , Blau H M
Published: May 2006

Antibodies to the acetylcholine receptor (AChR), which are diagnostic of the human autoimmune disease myasthenia gravis, block AChR function and increase the rate of AChR degradation leading to impaired neuromuscular transmission. Steroids are frequently used to alleviate symptoms of muscle fatigue and weakness in patients with myasthenia gravis because of their well-documented immunosuppressive effects. We show here that the steroid dexamethasone significantly increases total surface AChRs on cultured human muscle exposed to myasthenia gravis sera. Our results suggest that the clinical improvement observed in myasthenic patients treated with steroids is due not only to an effect on the immune system but also to a direct effect on muscle. We propose that the identification and development of pharmacologic agents that augment receptors and other proteins that are reduced by human genetic or autoimmune disease will have broad therapeutic applications.
Early folding intermediate of ribonuclease A.
Authors: Udgaonkar J B , Baldwin R L
Published: May 2006

Pulsed hydrogen exchange (2H-1H) is used to characterize the folding process of ribonuclease A (disulfide bonds intact). The results show one principal early folding intermediate (I1), which is formed rapidly after the start of folding and whose proton-exchange properties change with the time of folding. All probes that are hydrogen bonded within the beta-sheet of native ribonuclease A are protected in I1. Thus, the results suggest that the beta-sheet is formed rapidly and cooperatively. The initial protection factors of probes in the beta-sheet are between 10 and 100, but they increase with time of folding and exceed 1000 at 400 msec from the start of folding. Thus, the beta-sheet is only moderately stable when it is first formed, but subsequent events stabilize it, possibly through interactions involving hydrophobic side chains. The large protection factors of the beta-sheet probes in an early folding intermediate are unexpected and remarkable. Probes in the three alpha-helices are fewer in number and give less accurate data than the beta-strand probes. The folding kinetics expected for a simple sequential model of folding are outlined. An important difference between the observed and predicted behavior is that the early folding intermediate is not fully populated when it is first formed.
Acquisition of protease resistance by prion proteins in scrapie-infected cells does not require asparagine-linked glycosylation.
Authors: Taraboulos A , Rogers M , Borchelt D R , McKinley M P , Scott M , Serban D , Prusiner S B
Published: May 2006

The scrapie and cellular isoforms of the prion protein (PrPSc and PrPC) differ strikingly in a number of their biochemical and metabolic properties. The structural features underlying these differences are unknown, but they are thought to result from a posttranslational process. Both PrP isoforms contain complex type oligosaccharides, raising the possibility that differences in the asparagine-linked glycosylation account for the properties that distinguish PrPC and PrPSc. ScN2a and ScHaB cells in culture produce several PrP molecules with relative molecular masses of 26-35 kDa and proteinase K-resistant cores of 19-29 kDa. When the cells were treated with tunicamycin, this heterogeneity was eliminated and a single PrP species of 26 kDa was observed. Several hours after its synthesis, a fraction of this protein became insoluble in detergents and acquired a proteinase K-resistant core, thus displaying two of the biochemical hallmarks of PrPSc. Synthesis in the presence of tunicamycin restricted the proteinase K-resistant cores of PrP to a single species of 19 kDa. No proteinase K-resistant PrP was found in uninfected cells. Expression of a mutated PrP gene lacking both asparagine-linked glycosylation sites in ScN2a cells resulted in the synthesis of 19-kDa proteinase K-resistant PrP molecules. We conclude that asparagine-linked glycosylation is not essential for the synthesis of proteinase K-resistant PrP and that structural differences unrelated to asparagine-linked oligosaccharides must exist between PrPC and PrPSc. Whether unglycosylated PrPSc molecules are associated with scrapie prion infectivity remains to be established.
Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes.
Authors: Kozak M
Published: May 2006

Recognition of an AUG initiator codon in a suboptimal context improves when a modest amount of secondary structure is introduced near the beginning of the protein-coding sequence. This facilitating effect depends on the position of the downstream stem-loop (hairpin) structure. The strongest facilitation is seen when the hairpin is separated from the preceding AUG codon by 14 nucleotides. Because 14 nucleotides corresponds to the approximate distance between the leading edge of the ribosome and its AUG-recognition center as measured by ribonuclease protection experiments, a likely explanation for the enhancing effect of a downstream hairpin is that secondary structure slows scanning, thereby providing more time for recognition of the AUG codon, and the facilitation is greatest when the 40S ribosome stalls with its AUG-recognition center directly over the AUG. The variable ability of mammalian ribosomes to initiate at non-AUG codons in vitro is also explicable by the presence or absence of a stem-loop structure just downstream from the alternative initiator codon. This may be relevant to recent reports of adventitious upstream initiation events at non-AUG codons in some vertebrate mRNAs that have structure-prone, G + C-rich leader sequences.
Homeobox gene expression plus autocrine growth factor production elicits myeloid leukemia.
Authors: Perkins A , Kongsuwan K , Visvader J , Adams J M , Cory S
Published: May 2006

In the murine myelomonocytic leukemia WEHI-3B, proviral insertions have induced expression of the Hox-2.4 homeobox gene and the gene for the myeloid growth factor interleukin 3 (IL-3). To assess their potential oncogenic role, normal bone marrow cells were infected with retroviruses bearing the genes for IL-3 or IL-3 plus Hox-2.4. Unlike the IL-3 virus, the IL-3/Hox-2.4 virus was highly leukemogenic. Infected cells expressing both genes exhibited retarded differentiation in vitro, generated myelomonocytic cell lines, and provoked a rapid, transplantable myeloid leukemia in vivo. The oncogenic action of Hox-2.4 appears to derive from its ability to impede the IL-3-driven terminal differentiation of myeloid cells. The results suggest that homeobox genes can regulate key differentiation processes such as self-renewal capacity and that their inappropriate expression can be oncogenic.
cDNA cloning of a mouse mammary epithelial cell surface protein reveals the existence of epidermal growth factor-like domains linked to factor VIII-like sequences.
Authors: Stubbs J D , Lekutis C , Singer K L , Bui A , Yuzuki D , Srinivasan U , Parry G
Published: May 2006

A 2.1-kilobase cDNA coding for a surface protein of mammary epithelial cells has been isolated from a mouse mammary gland lambda gt11 cDNA library. Sequence analysis of this cDNA reveals an open reading frame of 1389 base pairs that defines a protein with a molecular mass of 51.5 kDa. Structural analysis of the predicted sequence identifies two putative functional domains of the protein: (i) an N-terminal cysteine-rich region that is similar to epidermal growth factor-like domains of Drosophila Notch-1 protein and (ii) a large segment of the sequence that exhibited 54.5% identity with C-terminal domains of human coagulation factors VIII and V. These similarities in structure are used to predict the possible functions of the protein and its means of interaction with the cell surface. mRNA expression was detectable in mammary tissue from nonpregnant animals but was maximal in the lactating gland. In cultured cells, mRNA levels also correlated with the degree of cellular differentiation.
The left spectrum, the Levitzki radical, and noncommutative schemes.
Authors: Rosenberg A L
Published: May 2006

This note contains a brief exposition of the basics of a noncommutative version of affine, quasi-affine, and projective algebraic geometry. In this version, to any associative ring (with unity) a quasi-affine (resp. affine) left scheme is assigned. The notion of the left spectrum of a ring plays the key role.
The metabolism of L-arginine and its significance for the biosynthesis of endothelium-derived relaxing factor: cultured endothelial cells recycle L-citrulline to L-arginine.
Authors: Hecker M , Sessa W C , Harris H J , Anggård E E , Vane J R
Published: May 2006

We have investigated the mechanism by which cultured endothelial cells generate L-arginine (L-Arg), the substrate for the biosynthesis of endothelium-derived relaxing factor. When Arg-depleted endothelial cells were incubated in Krebs' solution for 60 min, L-Arg levels were significantly (9.7-fold) elevated. The generation of L-Arg coincided with a substantial decrease (90%) in intracellular L-glutamine (L-Gln), whereas all other amino acids were virtually unaffected. Changes in calcium, pH, or oxygen tension had no effect on L-Arg generation, which was, however, prevented when the cells were incubated in culture medium containing L-Gln. L-Arg generated by endothelial cells labeled with L-[14C]Arg was derived from an unlabeled intracellular source, for the specific activity of the intracellular L-Arg pool decreased substantially (8.8-fold) over 60 min. Arg-depleted endothelial cells did not form urea or metabolize L-ornithine but converted L-citrulline (L-Cit) to L-Arg possibly via formation of L-argininosuccinic acid. Nondepleted cells stimulated with the calcium ionophore A23187 showed only a transient accumulation of L-Cit, indicating that L-Cit is recycled to L-Arg during the biosynthesis of endothelium-derived relaxing factor. The generation of L-Arg by Arg-depleted endothelial cells was partially (45%) blocked by protease inhibitors, and various Arg-containing dipeptides were rapidly cleaved to yield L-Arg. Thus, cultured endothelial cells recycle L-Cit to L-Arg and possibly liberate peptidyl L-Arg. The Arg-Cit cycle appears to be the equivalent in the endothelial cell to the formation of urea by the liver. The biosynthesis of endothelium-derived relaxing factor may, therefore, not only produce a powerful vasodilator but also relieve the endothelial cell of excess nitrogen.
Cold fusion in palladium: a more realistic calculation.
Authors: Parmenter R H , Lamb W E
Published: May 2006

The Thomas-Fermi-Mott equation is modified to take account of the fact that conduction electrons in a metal may be considered to have an effective mass at wave-numbers comparable with or less than the inverse Debye screening length, but they should be considered to have the free-electron mass at much larger wavenumbers. This modification allows for a more realistic calculation of the fusion rate of deuteron pairs in palladium, this rate being 10(-23) sec-1, comparable with some experimental results. The Oppenheimer-Phillips process enhances the rate by a factor of 2.262.
Analysis of gene families encoding acidic and basic beta-1,3-glucanases of tobacco.
Authors: Linthorst H J , Melchers L S , Mayer A , van Roekel J S , Cornelissen B J , Bol J F
Published: May 2006

Healthy tobacco plants accumulate beta-1,3-glucanases (glucan endo-1,3-beta-glucosidase; EC 3.2.1.39) in their roots and in specific parts of the flowers. After infection with tobacco mosaic virus, acidic and basic beta-1,3-glucanases are induced in the inoculated and virus-free leaves of the plant. An analysis of cDNA clones demonstrated that at least five genes for acidic beta-1,3-glucanases are induced after tobacco mosaic virus infection. Southern blot analysis indicated that the tobacco genome contains approximately eight genes for acidic beta-1,3-glucanases and a smaller number of genes encoding basic beta-1,3-glucanases. Genes from both gene families were cloned and sequenced. The basic isozymes contain a C-terminal extension that is cleaved off during their targeting to the vacuoles. This extension is absent in the acidic isozymes, which accumulate extracellularly. Northern blot hybridization showed that genes encoding acidic and basic beta-1,3-glucanases are strongly induced after tobacco mosaic virus infection or salicylate treatment of tobacco. The cloning of these genes is a first step toward the identification of regulatory elements involved in their coordinate induction.
Systemic movement of an RNA plant virus determined by a point substitution in a 5' leader sequence.
Authors: Petty I T , Edwards M C , Jackson A O
Published: May 2006

The ability of viruses to move through infected plants is an important determinant of host range and pathogenicity. We have investigated the genetic basis for the inability of the Type strain of barley stripe mosaic hordeivirus to undergo long-range systemic movement in the tobacco Nicotiana benthamiana. We show that, in this model system, a short open reading frame in the 5' leader of the smallest viral genomic RNA prevents long-range vascular movement. As predicted by the ribosome scanning model, the leader open reading frame decreases the efficiency with which the 5'-proximal gene is translated in vitro. Thus, systemic pathogenicity in this system may be determined by the efficiency of translation of a viral gene in vivo and is not determined by the primary sequence of the encoded protein.
Expression of the archaebacterial bacterio-opsin gene with and without signal sequences in Escherichia coli: the expressed proteins are located in the membrane but bind retinal poorly.
Authors: Karnik S , Doi T , Molday R , Khorana H G
Published: May 2006

In a further effort to obtain functional expression of the bacterio-opsin gene (bop) in Escherichia coli, the bop gene with E. coli signal sequences as well as the bop gene with the native presequence were expressed in E. coli. The location of the expressed products in the E. coli cell and their processing and folding to a structure that binds retinal as in Halobacterium halobium were investigated. All the expressed proteins were in the membrane. The proteins were largely unprocessed, and they were distributed between the outer and the inner membrane. The processed fractions, which were minor, were exclusively in the inner membrane. The processed proteins bound exogenously added all-trans-retinal but only partially, indicating that these proteins were present in at least two folded states.
Sequence-specific interaction of Tat protein and Tat peptides with the transactivation-responsive sequence element of human immunodeficiency virus type 1 in vitro.
Authors: Cordingley M G , LaFemina R L , Callahan P L , Condra J H , Sardana V V , Graham D J , Nguyen T M , LeGrow K , Gotlib L , Schlabach A J
Published: May 2006

Bacterially expressed Tat protein of human immunodeficiency virus type 1 binds selectively to short RNA transcripts containing the viral transactivation-responsive element (TAR). Sequences sufficient for Tat interaction map to the distal portion of the TAR stem-loop. We show that critical sequences for Tat binding are located in the single-stranded "bulge," but no requirement for specific "loop" sequences could be demonstrated. TAR RNA competed for complex formation, and TAR mutants exhibited up to 10-fold reduced affinity for Tat. Synthetic peptides containing the basic region of Tat bound selectively to TAR RNA and exhibited the same sequence requirements and similar relative affinities for mutant TAR RNA as the intact protein. These results suggest that Tat contains a small RNA-binding domain capable of recognizing TAR and implicate functional relevance for direct Tat-TAR interaction in transactivation.
Overexpression of normal and mutated forms of HRAS induces orthotopic bladder invasion in a human transitional cell carcinoma.
Authors: Theodorescu D , Cornil I , Fernandez B J , Kerbel R S
Published: May 2006

Recent studies have shown that orthotopic (transurethral) transplantation of human bladder cancer cell lines into nude mice permits tumor growth that accurately reflects their clinical malignant status in the original host. Thus, such a system allows a unique opportunity to analyze the genetic events involved in the conversion of low-grade bladder cancer, the vast majority of which are curable, to the high-grade life-threatening form of the disease. Since 5-10% of transitional cell carcinomas (TCCs) have been shown to contain a mutated HRAS gene, and protein expression levels of all forms of HRAS have been correlated with TCC progression, we chose to study the contribution of the HRAS oncogene in bladder tumor progression. We evaluated the effects of transfection of normal or mutated HRAS genes into a human TCC, called RT-4, that behaves as a superficial noninvasive papillary tumor after transurethral orthotopic inoculation into athymic nude mice. We found that overexpression of either transfected normal or mutated HRAS genes converted RT-4 cells to express an invasive phenotype remarkably similar in nature to the clinical behavior of high-grade bladder carcinomas. These results suggest a role for overexpressed normal or mutated RAS genes in human bladder carcinoma progression, and highlight the importance of using orthotopic inoculation systems for evaluation of the contribution of oncogenes to malignant tumor progression.
Identification of a phenylalkylamine binding region within the alpha 1 subunit of skeletal muscle Ca2+ channels.
Authors: Striessnig J , Glossmann H , Catterall W A
Published: May 2006

The alpha 1 subunit of the skeletal muscle Ca2+ channel has been specifically photoaffinity labeled with the phenylalkylamine-receptor-selective verapamil derivative (-)-5-(3-azidophenethyl[N-methyl-3H]methylamino)-2-(3,4,5- trimethoxyphenyl)-2-isopropylvaleronitrile ([N-methyl-3H]LU49888). Proteolytic fragments generated by various endoproteases were probed by immunoprecipitation with several sequence-specific antibodies to determine the site of labeling within the primary structure of alpha 1. These results restrict the site of photolabeling by [N-methyl-3H]LU49888 to the region between Glu-1349 and Trp-1391. This segment of alpha 1 contains transmembrane helix S6 of domain IV and the beginning of the long intracellular C-terminal tail. Because of the phenylalkylamine receptor site is only accessible from the intracellular side of the Ca2+ channel, we propose that the intracellular end of helix IVS6 and the adjacent intracellular amino acid residues play an essential role in formation of the phenylalkylamine receptor site. The action of the phenylalkylamines as open-channel blockers suggests that this region may also contribute to formation of the intracellular opening of the transmembrane pore of the Ca2+ channel.
Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum.
Authors: Shak S , Capon D J , Hellmiss R , Marsters S A , Baker C L
Published: May 2006

Respiratory distress and progressive lung destruction in cystic fibrosis can be attributed to bacterial persistence and the accumulation of viscous purulent secretions in the airways. More than 30 yr ago it was suggested that the large amounts of DNA in purulent secretions contribute to its viscosity and that bovine pancreatic DNase I could reduce the viscosity. To evaluate the potential clinical utility of recombinant human DNase I (rhDNase) in the treatment of cystic fibrosis, we have cloned, sequenced, and expressed rhDNase. Catalytic amounts of rhDNase greatly reduce the viscosity of purulent cystic fibrosis sputum, transforming it within minutes from a nonflowing viscous gel to a flowing liquid. The reduction in viscosity is associated with a decrease in size of DNA in the sputum. Inhalation of a rhDNase aerosol may be a simple direct approach that will help individuals with cystic fibrosis and other patients with pneumonia or bronchitis to clear their airways of purulent secretions.
Immunological evidence for the accumulation of lipoprotein(a) in the atherosclerotic lesion of the hypoascorbemic guinea pig.
Authors: Rath M , Pauling L
Published: May 2006

Lipoprotein(a) [Lp(a)] is an extremely atherogenic lipoprotein. Lp(a) has been found in the plasma of humans and other primates, but until now only in a few other species. The mechanism by which it exerts its atherogenicity is still poorly understood. We observed that Lp(a) has been found in the plasma of several species unable to synthesize ascorbate and not in other species. We have now detected apoprotein(a) in the plasma of the guinea pig. We induced atherosclerosis in this animal by dietary ascorbate depletion and, using SDS/PAGE and subsequent immunoblotting, we identified Lp(a) as accumulating in the atherosclerotic plaque. Most importantly, adequate amounts of ascorbate (40 mg per kg of body weight per day) prevent the development of atherosclerotic lesions in this animal model and the accumulation of Lp(a) in the arterial wall. We suggest an analogous mechanism in humans because of the similarity between guinea pigs and humans with respect to both the lack of endogenous ascorbate production and the role of Lp(a) in human atherosclerosis.
Effects of glycyl-L-glutamine and methylprednisolone on maintenance of acetylcholinesterase of transected rat sciatic nerves.
Authors: Koelle G B , Han M S
Published: May 2006

Under anesthesia with sodium pentobarbital, the sciatic nerves of rats were transected bilaterally, and a catheter was inserted into the central end of the left renal artery. After an initial flush, an Alzet pump was attached to the catheter, containing various concentrations of glycyl-L-glutamine (Gly-Gln), methylprednisolone sodium succinate (MePred), or both. Rats were sacrificed at intervals of 2, 4, or 6 days; the peripheral portions of the sciatic nerves were excised, homogenized, and centrifuged, and the supernates were assayed for acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7) and protein. Significantly higher contents of AcChoEase over untreated transected controls were obtained (i) at 4 days posttransection in rats infused with 0.015 M Gly-Gln and (ii) at 6 days posttransection in rats infused with MePred at 3.0 mg.kg-1.hr-1 after an initial dose of 120 mg/kg with or without Gly-Gln.
Electrostatic field around cytochrome c: theory and energy transfer experiment.
Authors: Northrup S H , Wensel T G , Meares C F , Wendoloski J J , Matthew J B
Published: May 2006

Energy transfer in the "rapid diffusion" limit from electronically excited terbium(III) chelates in three different charge states to horse heart ferricytochrome c was measured as a function of ionic strength. Theoretical rate constants calculated by numerical integration of the Forster integral (containing the Poisson-Boltzmann-generated protein electrostatic potential) were compared with the experimental data to evaluate the accuracy of protein electrostatic field calculations at the protein/solvent interface. Two dielectric formalisms were used: a simple coulombic/Debye-Hückel procedure and a finite difference method [Warwicker, J. & Watson, H. C. (1982) J. Mol. Biol. 157, 671-679] that accounts for the low-dielectric protein interior and the irregular protein/solvent boundary. Good agreement with experiment was obtained and the ionic-strength dependence of the reaction was successfully reproduced. The sensitivity of theoretical rate constants to the choices of effective donor sphere size and the energy transfer distance criterion was analyzed. Electrostatic potential and rate-constant calculations were carried out on sets of structures collected along two molecular dynamics trajectories of cytochrome c. Protein conformational fluctuations were shown to produce large variations in the calculated energy transfer rate constant. We conclude that protein fluctuations and the resulting transient structures can play significant roles in biological or catalytic activities that are not apparent from examination of a static structure. For calculating protein electrostatics, large-scale low-frequency conformational fluctuations, such as charged side-chain reorientation, are established to be as important as the computational method for incorporating dielectric boundary effects.
Molecular mechanisms in down-regulation of tumor necrosis factor expression.
Authors: Haas J G , Baeuerle P A , Riethmüller G , Ziegler-Heitbrock H W
Published: May 2006

Excessive production of tumor necrosis factor (TNF) after stimulation by lipopolysaccharide (LPS) may result in fever, intravascular coagulation, and lethal shock. An efficient way of preventing the excessive TNF production is desensitization of monocytes/macrophages to LPS. We have analyzed the molecular mechanisms involved in the induction of desensitization and the mechanisms operative in the desensitized, LPS-refractory cells by employing the human monocytic cell line Mono-Mac-6. Similar to human blood monocytes, treatment of Mono-Mac-6 cells with LPS (1 microgram/ml) results in a rapid and transient expression of TNF. When Mono-Mac-6 cells are precultured in medium containing low levels of LPS, they become refractory to subsequent LPS stimulation and show no or little secretion of TNF protein. Desensitization can be blocked by the inhibition of cyclooxygenase and protein kinase C; both prostaglandin E2 (together with a second signal) and phorbol 12-myristate 13-acetate can mimic desensitization. By employing prostaglandin E2 and low concentrations of phorbol 12-myristate 13-acetate, a synergism in the induction of desensitization can be demonstrated. Hence, our studies show that two distinct pathways are involved in the induction of hyporesponsiveness. In both LPS-responsive and LPS-desensitized Mono-Mac-6 cells, LPS was able to induce the transcription factor NF-kappa B in the nucleus. Still, the prevalence of TNF-specific mRNA was dramatically reduced in the desensitized cells. These data indicate that LPS-desensitized Mono-Mac-6 cells are able to activate initial steps of signal transduction up to the level of the NF-kappa B transcription factor. The absence of TNF transcripts, however, indicates that additional nuclear factors may be missing or that silencers may be active such that transcription of the TNF gene is prevented.
Invasion resistance arises in strongly interacting species-rich model competition communities.
Authors: Case T J
Published: May 2006

I assemble stable multispecies Lotka-Volterra competition communities that differ in resident species number and average strength (and variance) of species interactions. These are then invaded with randomly constructed invaders drawn from the same distribution as the residents. The invasion success rate and the fate of the residents are determined as a function of community-and species-level properties. I show that the probability of colonization success for an invader decreases with community size and the average strength of competition (alpha). Communities composed of many strongly interacting species limit the invasion possibilities of most similar species. These communities, even for a superior invading competitor, set up a sort of "activation barrier" that repels invaders when they invade at low numbers. This "priority effect" for residents is not assumed a priori in my description for the individual population dynamics of these species; rather it emerges because species-rich and strongly interacting species sets have alternative stable states that tend to disfavor species at low densities. These models point to community-level rather than invader-level properties as the strongest determinant of differences in invasion success. The probability of extinction for a resident species increases with community size, and the probability of successful colonization by the invader decreases. Thus an equilibrium community size results wherein the probability of a resident species' extinction just balances the probability of an invader's addition. Given the distribution of alpha it is now possible to predict the equilibrium species number. The results provide a logical framework for an island-biogeographic theory in which species turnover is low even in the face of persistent invasions and for the protection of fragile native species from invading exotics.
Molecular analysis of human argininosuccinate lyase: mutant characterization and alternative splicing of the coding region.
Authors: Walker D C , McCloskey D A , Simard L R , McInnes R R
Published: May 2006

Argininosuccinic acid lyase (ASAL) deficiency is a clinically heterogeneous autosomal recessive urea cycle disorder. We previously established by complementation analysis that 28 ASAL-deficient patients have heterogeneous mutations in a single gene. To prove that the ASAL structural gene is the affected locus, we sequenced polymerase chain reaction-amplified ASAL cDNA of a representative mutant from the single complementation group. Fibroblast strain 944 (approximately 1% of residual ASAL activity), from a late-onset patient who was the product of a consanguineous mating, had only a single base-pair change in the coding region, a C-283----T transition at a CpG dinucleotide in exon 3. This substitution converts Arg-95 to Cys (R95C), occurs in a stretch of 13 residues that is identical in yeast and human ASAL, and was present in both of the patient's alleles but not in 14 other mutant or 10 normal alleles. Expression in COS cells demonstrated that the R95C mutation produces normal amounts of ASAL mRNA but little protein and less than 1% ASAL activity. We observed that amplified cDNA from mutant 944 and normal cells (liver, keratinocytes, lymphoblasts, and fibroblasts) contained, in addition to the expected 5' 513-base-pair band, a prominent 318-base-pair ASAL band formed by the splicing of exon 2 from the transcript. The short transcript maintains the ASAL reading frame but removes Lys-51, a residue that may be essential for catalysis, since it binds the argininosuccinate substrate. We conclude (i) that the identification of the R95C mutation in strain 944 demonstrates that virtually all ASAL deficiency results from defects in the ASAL structural gene and (ii) that minor alternative splicing of the coding region occurs at the ASAL locus.
Variation in repeated nucleotide sequences sheds light on the phylogeny of the wheat B and G genomes.
Authors: Dvorák J , Zhang H B
Published: May 2006

A general method based on variation in repeated nucleotide sequences was developed for the identification of diploid species most closely related to a specific genome of a polyploid species. The utility of this method was demonstrated by showing that Triticum speltoides is the most closely related extant species to both the B and G genomes of tetraploid wheats.
Cross-linked A alpha.gamma chain hybrids serve as unique markers for fibrinogen polymerized by tissue transglutaminase.
Authors: Murthy S N , Lorand L
Published: May 2006

Notwithstanding the high degree of amino acid sequence homologies between human factor XIIIa on the one hand and intracellular transglutaminases (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) from guinea pig liver or human erythrocytes on the other, we find that the two sets of enzymes differ remarkably in the mode of cross-linking the same protein substrate--i.e., human fibrinogen. In the program of polymerization with factor XIIIa, production of the known gamma-gamma' homologous chain pairs is the dominant feature, whereas with either intracellular transglutaminase, a series of hitherto unidentified A alpha.gamma hybrid chain combinations, designated A alpha p gamma q (p and q = 1, 2, 3...), is generated and practically no gamma-gamma' dimers are formed. Two-dimensional electrophoresis is particularly useful for demonstrating the production of A alpha p gamma q structures by protein staining as well as by immunoblotting against specific antibodies to the A alpha and gamma chains of fibrinogen. These findings should aid in deciding whether the direct cross-linking of fibrinogen by transglutaminase might contribute to thrombotic processes in addition to the thrombin- and factor XIIIa-dependent pathway of clot formation.
Involvement of transforming growth factor alpha in the release of luteinizing hormone-releasing hormone from the developing female hypothalamus.
Authors: Ojeda S R , Urbanski H F , Costa M E , Hill D F , Moholt-Siebert M
Published: May 2006

Little is known about the presence of trophic factors in the hypothalamus and the role they may play in regulating the functional development of hypothalamic neurons. We have investigated the ability of epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) to affect the release of luteinizing hormone-releasing hormone (LHRH), the neuropeptide that controls reproductive development. We have also determined whether the genes encoding EGF and TGF-alpha are expressed in the prepubertal female hypothalamus. Northern blot analysis of poly(A)+ RNA utilizing a single-stranded EGF cDNA probe failed to reveal the presence of EGF mRNA in either the hypothalamus or the cerebral cortex at any age studied (fetal day 18 to postnatal day 36). In contrast, both a complementary RNA probe and a double-stranded TGF-alpha cDNA recognized in these regions a 4.5-kilobase (kb) mRNA species identical to TGF-alpha mRNA. The abundance of TGF-alpha mRNA was 3-4 times greater in the hypothalamus than in the cerebral cortex. Both EGF and TGF-alpha (2-100 ng/ml) elicited a dose-related increase in LHRH release from the median eminence of juvenile rats in vitro. They also enhanced prostaglandin E2 (PGE2) release. The transforming growth factors TGF-beta 1 and -beta 2 were ineffective. Only a high dose of basic fibroblast growth factor was able to increase LHRH and PGE2 release. Blockade of the EGF receptor transduction mechanism with RG 50864, a selective inhibitor of EGF receptor tyrosine kinase activity, prevented the effect of both EGF and TGF-alpha on LHRH and PGE2 release but failed to inhibit the stimulatory effect of PGE2 on LHRH release. Inhibition of prostaglandin synthesis abolished the effect of TGF-alpha on LHRH, indicating that PGE2 mediates TGF-alpha-induced LHRH release. The results indicate that the effect of EGF and TGF-alpha on LHRH release is mediated by the EGF/TGF-alpha receptor and suggest that TGF-alpha rather than EGF may be the physiological ligand for this interaction. Since in the central nervous system most EGF/TGF-alpha receptors are located on glial cells, the results also raise the possibility that--at the median eminence--TGF-alpha action may involve a glial-neuronal interaction, a mechanism by which the trophic factor first stimulates PGE2 release from glial cells, and then PGE2 elicits LHRH from the neuronal terminals.
Steroidogenic activity of a peptide specified by the reversed sequence of corticotropin mRNA.
Authors: Clarke B L , Blalock J E
Published: May 2006

The molecular recognition theory predicts that a reversed (3'----5') reading of an mRNA should yield a peptide that is structurally and functionally similar to that specified in the 5'----3' direction. We tested this idea by synthesizing a corticotropin (ACTH) analogue using a reverse reading of bovine mRNA for ACTH-(1-24). This peptide, designated ACTH-3'----5', had a similar hydropathic profile to native ACTH-5'----3' but had only 30% sequence homology and eight different charge substitutions. ACTH-3'----5' specifically bound to the surface of mouse Y-1 adrenal cells and to polyclonal anti-ACTH antibody. Additionally, ACTH-3'----5' stimulated cAMP synthesis and steroidogenesis in adrenal cells. These findings show that ACTH-3'----5' mimics the corticotropic properties of native ACTH, thereby further validating the molecular recognition theory.