bluuebunny/crossref_metadata_2025 huggingface dataset:
NMR detection of creatine kinase expressed in liver of transgenic mice: determination of free ADP levels.
Authors: Koretsky A P , Brosnan M J , Chen L H , Chen J D , Van Dyke T
Published: May 2006

To use the equilibrium established by creatine kinase (CK) to determine hepatic free ADP levels, the transcriptional control elements of the transthyretin gene were used to direct expression of the CK B isozyme to the livers of transgenic mice. Activities of CK ranging from 80-250 mumol per min per g (wet weight) were detected in liver extracts from five founder mice. The CK activity was stably transmitted to subsequent generations. Isozyme gels and immunoblots confirmed that the activity detected in extracts was due to the B isozyme of CK. Immunohistology indicated that the protein was expressed uniformly throughout the liver and was localized primarily to the cytoplasm. 31P NMR spectroscopy was used to detect the metabolic product of the CK reaction, phosphocreatine, demonstrating that the enzyme was active in vivo. The phosphocreatine level fell rapidly during anoxia (t1/2 = 1 min), indicating that the CK reaction was integrated into hepatic energy metabolism. The equilibrium established by CK was used to calculate a hepatic free ADP level of 0.059 +/- 0.004 mumol/g (wet weight). In vivo NMR studies of these mice will be valuable for studying the role of free ADP in regulating liver metabolism.
Opioid and nicotine receptors affect growth regulation of human lung cancer cell lines.
Authors: Maneckjee R , Minna J D
Published: May 2006

Using specific ligands, we find that lung cancer cell lines of diverse histologic types express multiple, high-affinity (Kd = 10(-9)-10(-10) M) membrane receptors for mu, delta, and kappa opioid agonists and for nicotine and alpha-bungarotoxin. These receptors are biologically active because cAMP levels decreased in lung cancer cells after opioid and nicotine application. Nicotine at concentrations (approximately 100 nM) found in the blood of smokers had no effect on in vitro lung cancer cell growth, whereas mu, delta, and kappa opioid agonists at low concentrations (1-100 nM) inhibited lung cancer growth in vitro. We also found that lung cancer cells expressed various combinations of immunoreactive opioid peptides (beta-endorphin, enkephalin, or dynorphin), suggesting the participation of opioids in a negative autocrine loop or tumor-suppressing system. Due to the almost universal exposure of patients with lung cancer to nicotine, we tested whether nicotine affected the response of lung cancer cell growth to opioids and found that nicotine at concentrations of 100-200 nM partially or totally reversed opioid-induced growth inhibition in 9/14 lung cancer cell lines. These in vitro results for lung cancer cells suggest that opioids could function as part of a "tumor suppressor" system and that nicotine can function to circumvent this system in the pathogenesis of lung cancer.
Transcriptional effects of polyamines on ribosomal proteins and on polyamine-synthesizing enzymes in Escherichia coli.
Authors: Huang S C , Panagiotidis C A , Canellakis E S
Published: May 2006

We find that the transcription of various ribosomal proteins can be differentially affected by polyamines and by changes in growth rates. Using strain MG1655 of Escherichia coli K-12 (F-, lambda-), we have determined the effects of polyamines and changes in growth rate on the transcription of several ribosomal genes and the polyamine-synthesizing enzymes ornithine decarboxylase (L-ornithine carboxy-lyase; EC 4.1.1.17) and arginine decarboxylase (L-arginine carboxylyase; EC 4.1.1.19). Ribosomal proteins S20 and L34 can be differentiated from the other ribosomal proteins studied; the transcription of S20 and L34 is especially sensitive to polyamines and less sensitive to changes in growth rates. In contrast, the transcription of S10, S15, S19, L2, L4, L20, L22, and L23 is insensitive to polyamines although it is particularly sensitive to changes in growth rates. Like S20 and L34, the transcription of ornithine decarboxylase and arginine decarboxylase is especially sensitive to polyamines. Polyamines specifically enhance the transcription of ribosomal proteins S20 and L34, and decrease that of ornithine decarboxylase and arginine decarboxylase. It is evident that polyamines can exert both positive and negative regulation of gene expression in E. coli that can be differentiated from the effects caused by changes in growth rates.
Evidence that a distribution of bacterial reaction centers underlies the temperature and detection-wavelength dependence of the rates of the primary electron-transfer reactions.
Authors: Kirmaier C , Holten D
Published: May 2006

The rates of the primary electron-transfer processes in Rhodobacter sphaeroides reaction centers have been examined in detail by using 150-fs excitation flashes at 870 nm. At room temperature the apparent time constants for both initial charge separation (P* --> P+BPhL-) and subsequent electron transfer (P+BPhL- --> P+QA-) are found to encompass a range of values (approximately 1.3-4 ps and approximately 100-320 ps, respectively), depending on the wavelength at which the kinetics are followed. We suggest this reflects a distribution of reaction centers (or a few conformers), having differences in factors such as distances or orientations between the cofactors, hydrogen bonding, or other pigment-protein interactions. We also suggest that the time constants observed at cryogenic temperatures (approximately 1.3 and approximately 100 ps, respectively, with much smaller or negligible variation with detection wavelength) do not reflect an actual increase in the rates with decreasing temperature but rather derive from a shift in the distribution of reaction centers toward those in which electron transfer inherently occurs with the faster rates.
Fibronectin controls capillary endothelial cell growth by modulating cell shape.
Authors: Ingber D E
Published: May 2006

An in vitro system has been developed to study the mechanism by which fibronectin (FN) regulates capillary endothelial cell growth in the presence of soluble angiogenic mitogens. Endothelial cells were cultured in chemically defined medium containing a constant, saturating amount of basic fibroblast growth factor. Formation of cell-FN contacts was then varied in a controlled fashion by three different techniques: (i) nonadhesive, bacteriological dishes were precoated with increasing densities of FN; (ii) soluble RGD peptides were used to progressively inhibit binding of cell-surface integrin receptors to adsorbed FN; and (iii) FN-coated surfaces were covered with increasingly thick layers of polyhydroxyethylmethacrylate (a nonadhesive polymer) to physically restrict cell access to FN binding sites. Endothelial cells became more extended and proliferated more rapidly as FN coating concentrations were raised from approximately 250 to approximately 10,000 FN molecules per micron 2. Computerized morphometric analysis confirmed that cell shape (projected cell areas) was determined by the density of FN contacts and that DNA synthetic levels were tightly coupled to the extent of cell spreading, regardless of the method used to perturb cell adhesion. In contrast, neither soluble FN nor cell-surface binding of FN-coated microbeads (diameter, 4.5 microns) had any effect on growth when cells were grown in suspension and cell spreading was prohibited. These results suggest that FN controls capillary endothelial cell proliferation based on its ability to support tension-dependent alterations of cell shape--i.e., both by binding to cell-surface integrins and by resisting mechanical loads that are applied to these receptors.
Structural basis for differential binding of staphylococcal enterotoxin A and toxic shock syndrome toxin 1 to class II major histocompatibility molecules.
Authors: Pontzer C H , Russell J K , Johnson H M
Published: May 2006

The related staphylococcal toxins staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin 1 (TSST-1) are microbial superantigens. They require interaction with class II major histocompatibility complex (MHC) molecules to activate T cells. We have previously identified a binding site on SEA, the N-terminal 45 amino acids, as well as its corresponding receptor on the MHC antigen, residues 65-85 of the beta chain. To further characterize the structural basis for SEA binding to class II MHC molecules we have examined its relationship to TSST-1 binding. Both toxins bound similarly to murine A20 cells, but blockage of binding was observed only with the homologous toxin, which suggests that the binding sites for the two toxins on A20 cells are distinct. In contrast, specific binding of SEA was greater than that of TSST-1 on human Raji cells. Further, SEA was a better inhibitor of TSST-1 binding than was TSST-1 itself at low concentrations, but TSST-1 only minimally inhibited SEA binding. The data suggest that TSST-1 interacts with Raji cells at an SEA binding site, but with a lower affinity. The peptides SEA-(1-45) and I-A beta b-(65-85) were capable of blocking SEA binding on both A20 and Raji cells, but blockage was more effective on A20 cells. Neither peptide was capable of blocking TSST-1 binding on either cell line. The data are compatible with a model in which SEA has a binding site on A20 cells involving SEA-(1-45) and I-A beta b-(65-85) which is distinct from that which binds TSST-1, while at least two binding sites are present on Raji cells. One site involves predominantly the residue 1-45 region on SEA and the 65-85 region of the MHC beta chain, while the other site involves both a different region on the SEA molecule and a different site on the class II MHC molecule to which it binds. This latter site also binds TSST-1.
Epidermal growth factor replaces estrogen in the stimulation of female genital-tract growth and differentiation.
Authors: Nelson K G , Takahashi T , Bossert N L , Walmer D K , McLachlan J A
Published: May 2006

The in vivo studies presented here demonstrate that epidermal growth factor (EGF) is an important autocrine and/or paracrine mediator of estrogen-induced growth and differentiation in mouse uterus and vagina. An antibody specific for EGF significantly inhibited estrogen-induced uterine and vaginal growth, thereby implicating EGF involvement in estrogen action. Furthermore, EGF administered via slow-release pellets in ovariectomized mice acted as a potent uterine and vaginal mitogen as well as an inducer of vaginal keratinization. Experiments with ovariectomized, adrenalectomized, hypophysectomized mice indicated that EGF mitogenesis does not require pituitary or adrenal hormones. Treatment with EGF also mimicked estrogen in the induction of uterine lactoferrin (a major estrogen-inducible secretory protein) mRNA and protein. These data suggest that EGF has estrogen-like effects in the promotion of cell growth in the reproductive tract and that EGF may serve as an important mediator of estrogen action in vivo.
Membrane-binding domain of the small G protein G25K contains an S-(all-trans-geranylgeranyl)cysteine methyl ester at its carboxyl terminus.
Authors: Yamane H K , Farnsworth C C , Xie H Y , Evans T , Howald W N , Gelb M H , Glomset J A , Clarke S , Fung B K
Published: May 2006

We showed previously that a 23-kDa guanine nucleotide-binding protein (G protein) purified from bovine brain membranes is carboxyl methylated and that this modification occurs at or near the membrane-binding domain. In the present study, we identified this small G protein as G25K (formerly termed Gp). We demonstrated that proteolytic digests of 3H-methylated G25K contained radiolabeled material that coeluted with synthetic S-(geranylgeranyl)cysteine methyl ester on reversed-phase HPLC. Further treatment by performic acid oxidation yielded radiolabeled material that coeluted with L-cysteic acid methyl ester, verifying that the isoprenoid moiety and carboxyl methyl ester are localized on a C-terminal cysteine residue. Analysis by gas chromatography-coupled mass spectrometry of material released from purified G25K by Raney nickel treatment positively identified the covalently bound lipid as an all-trans-geranylgeranyl (C20) isoprenoid moiety. These results suggest that geranylgeranyl modification and perhaps methyl esterification function in the membrane localization of this small G protein.
Sequence and functional expression in Xenopus oocytes of a human insulinoma and islet potassium channel.
Authors: Philipson L H , Hice R E , Schaefer K , LaMendola J , Bell G I , Nelson D J , Steiner D F
Published: May 2006

Regulation of insulin secretion involves the coordinated control of ion channels in the beta-cell membrane. We have isolated and characterized cDNA and genomic clones encoding a voltage-dependent K+ channel isoform expressed in human islets and in a human insulinoma. This K+ channel isoform, designated hPCN1, with a deduced amino acid sequence of 613 residues (Mr = 67,097), is related to the Shaker family of Drosophila K+ channels. hPCN1 is homologous to two other human K+ channel isoforms we have isolated, hPCN2 and hPCN3, with 55% and 65% amino acid sequence identity, respectively. The electrophysiological characteristics of hPCN1 were determined after microinjection of synthetic RNA into Xenopus oocytes. Two-microelectrode voltage-clamp recordings of oocytes injected with hPCN1 RNA revealed a voltage-dependent outward K+ current that inactivated slowly with time. Outward currents were inhibited by 4-aminopyridine with a Ki less than 0.10 mM and were relatively insensitive to tetraethylammonium ion or Ba2+. A delayed rectifier K+ channel such as hPCN1 could restore the resting membrane potential of beta cells after depolarization and thereby contribute to the regulation of insulin secretion.
Dimerization of mammalian progesterone receptors occurs in the absence of DNA and is related to the release of the 90-kDa heat shock protein.
Authors: DeMarzo A M , Beck C A , Onate S A , Edwards D P
Published: May 2006

In this study we have demonstrated that dimerization of mammalian progesterone receptors (PR) occurs in the absence of DNA. A specific immune coisolation assay was performed on extracts of T-47D human breast cancer cells with a monoclonal antibody specific for the full-length B form of progesterone receptor (PR-B). This resulted in coisolation of significant amounts of truncated form-A receptors (PR-A), indicating the presence of stable PR-A.PR-B dimers in solution. A positive correlation was observed between the ability of different receptor forms to oligomerize in solution and their ability to bind to specific DNA sequences. The ability to form stable PR-A.PR-B oligomers in the absence of DNA was also found to correlate with release of 90-kDa heat shock protein (hsp90) from the unactivated PR complex. These results support the hypothesis that dimerization in the absence of DNA is an important mechanism controlling receptor DNA-binding function and that hsp90 release may be a key step regulating dimerization. This suggests that hsp90 may function to repress DNA-binding activity indirectly by blocking receptor dimerization.
Expression from the murine p53 promoter is mediated by factor binding to a downstream helix-loop-helix recognition motif.
Authors: Ronen D , Rotter V , Reisman D
Published: May 2006

Expression of the p53 gene plays an important role in the regulation of cellular proliferation and malignant transformation. Overexpression of mutant forms of p53 is in fact a common feature of many transformed cells. Studies dealing with the transcriptional regulatory regions of the p53 gene indicate that, unlike most promoters transcribed by RNA polymerase II, the p53 promoter contains no TATA-like sequence upstream of the transcription start site. Here we demonstrate that the murine p53 promoter contains a cis-acting element that maps downstream to the transcription initiation site. The integrity of this element is required for high-level expression from the promoter in transformed cells. By DNase I protection and mobility-shift analysis, we show that a nuclear factor binds to this downstream element through the consensus recognition sequence for the helix-loop-helix (HLH)-containing proteins of the myc/MyoD family of transcriptional regulators. We propose that the activity of one or more members of this family of transcription factors is an important determinant in the expression of p53 and that at least one level of p53 overexpression in transformed cells may thus be due to aberrant expression of the relevant factor(s). Furthermore, the possibility that the regulation of expression of p53 occurs, in part, by means of a potential HLH-containing factor provides a possible mechanism for the suppression of proliferation by the MyoD family of transcriptional regulators.
Phenotypical characterization of the rat striatal neurons expressing the D1 dopamine receptor gene.
Authors: Le Moine C , Normand E , Bloch B
Published: May 2006

In situ hybridization experiments were performed in rat brain sections from normal and 6-hydroxydopamine-treated rats in order to map and identify the neurons expressing the D1 receptor gene in the striatum and the substantia nigra. Procedures of combined in situ hybridization, allowing the simultaneous detection of two mRNAs in the same section or in adjacent sections, were used to characterize the phenotypes of the neurons expressing the D1 receptor gene. D1 receptor mRNA was found in neurons all over the caudate-putamen, the accumbens nucleus, and the olfactory tubercle but not in the substantia nigra. In the caudate-putamen and accumbens nucleus, most of the neurons containing D1 receptor mRNA were characterized as medium-sized substance P neurons and distinct from those containing D2 receptor mRNA. Nevertheless, 15-20% of the substance P neurons did not contain D1 receptor mRNA. The neurons containing preproenkephalin A mRNA did not contain D1 receptor mRNA but contained D2 receptor mRNA. A small number of cholinergic and somatostatinergic neurons exhibited a weak reaction for D1 receptor mRNA. These results demonstrate that dopamine acts on efferent striatal neurons through expression of distinct receptors--namely, D1 and D2 in separate cell populations (substance P and preproenkephalin A neurons, respectively)--and can also act on nonprojecting neurons through D1 receptor expression.
A multimember kinesin gene family in Drosophila.
Authors: Endow S A , Hatsumi M
Published: May 2006

Degenerate primers to the kinesin motor domain were used in the polymerase chain reaction to amplify DNA sequences from Drosophila genomic DNA and cDNA libraries. The amplified DNA sequences were hybridized to polytene chromosomes and the map positions of the hybridizing sites were determined. More than 30 sites of hybridization were detected, indicating that the kinesin gene family may be much larger than previously thought. One new family member has already been identified as a result of this screen. The map positions should aid in the identification of further kinesin family members. Some of these kinesin-related genes are anticipated to function in previously undiscovered roles in the cell.
Intraspecific variation in population gene diversity and effective population size correlates with the mating system in plants.
Authors: Schoen D J , Brown A H
Published: May 2006

Published data on allele frequencies at isozyme loci in inbreeding and outbreeding plant species were analyzed to examine intraspecific variation in gene diversity and effective population size (Ne). Compared with outbreeders, inbreeding species showed markedly greater variation among populations in average values of Nei's gene diversity statistic. Effective population size was estimated by assuming that the variation observed at isozyme loci is selectively neutral. Inbreeding species showed greater levels of variation in Ne than did outbreeders, although the upper range of Ne was similar in the two classes of species. The results suggest that there may be considerable genetic variation and potential for evolutionary change in some but not all populations of inbreeders. Moreover, these findings are important with respect to the conservation of genetic resources. In particular, that the amount of intraspecific variation in population genetic diversity and Ne differs between inbreeding and outbreeding species should be taken into account in sampling efforts designed to optimize the diversity of germplasm collections.
Galanin: a hypothalamic-hypophysiotropic hormone modulating reproductive functions.
Authors: López F J , Merchenthaler I , Ching M , Wisniewski M G , Negro-Vilar A
Published: May 2006

Galanin (GAL) is widely distributed in the peripheral and the central nervous systems. In the brain, the highest GAL concentrations are observed within the hypothalamus and, particularly, in nerve terminals of the median eminence. This location, as well as GAL actions on prolactin, growth hormone, luteinizing hormone (LH), and LH-releasing hormone (LHRH) secretion, suggest the possibility that GAL may act as a putative hypothalamic-hypophysiotropic hormone. To establish this, GAL and LHRH levels were measured in hypophyseal portal plasma samples using specific radioimmunoassays. Rat galanin (rGAL) concentrations in portal blood were approximately 7-fold higher than those observed in peripheral plasma in male and female (estrus, diestrus) rats, indicating an active secretory process of rGAL into the portal vasculature. Frequent (10 min) sampling revealed that rGAL and LHRH were secreted into the portal circulation in a pulsatile manner with a pulse frequency of one pulse per hour. Interestingly, both hormone series depicted a high degree of coincident episodes. In fact, the probability of random coincidence, calculated by the algorithm HYPERGEO, was less than 0.01. Moreover, the retrograde tracer Fluoro-Gold, when given systemically, was taken up by GAL neurons in the hypothalamus, including a subset of neurons expressing rGAL and LHRH, strengthening the notion of the existence of a GAL neuronal system connected to the hypophyseal portal circulation. These observations reinforce the concept that GAL regulates pituitary hormone secretion. To analyze this in further detail, the effects of rGAL on LH secretion were evaluated under basal and stimulated conditions. rGAL induced a small but dose-dependent increase in LH secretion from cultured, dispersed pituitary cells. Interestingly, rGAL enhanced the ability of LHRH to stimulate LH release. The tight link between GAL and LHRH neuronal systems is strengthened by the observation that during the estrous cycle of the rat, rGAL and LHRH contents in the median eminence show an identical profile (r = 1.00). These data indicate that GAL should be considered as a hypothalamic-hypophysiotropic hormone and as an important neuromodulator of LHRH secretion and action. The colocalization and cosecretion of GAL and LHRH and the cooperative action at the level of the anterior pituitary afford important evidence for the functional significance of coexistence of neurotransmitters in neurons of the central nervous system.
Nitric oxide: an endogenous modulator of leukocyte adhesion.
Authors: Kubes P , Suzuki M , Granger D N
Published: May 2006

The objective of this study was to determine whether endogenous nitric oxide (NO) inhibits leukocyte adhesion to vascular endothelium. This was accomplished by superfusing a cat mesenteric preparation with inhibitors of NO production, NG-monomethyl-L-arginine (L-NMMA) or NG-nitro-L-arginine methyl ester (L-NAME), and observing single (30-microns diameter) venules by intravital video microscopy. Thirty minutes into the superfusion period the number of adherent and emigrated leukocytes, the erythrocyte velocity, and the venular diameter were measured; venular blood flow and shear rate were calculated from the measured parameters. The contribution of the leukocyte adhesion glycoprotein CD11/CD18 was determined using the CD18-specific monoclonal antibody IB4. Both inhibitors of NO production increased leukocyte adherence more than 15-fold. Leukocyte emigration was also enhanced, whereas venular shear rate was reduced by nearly half. Antibody IB4 abolished the leukocyte adhesion induced by L-NMMA and L-NAME. Incubation of isolated cat neutrophils with L-NMMA, but not L-NAME, resulted in direct upregulation of CD11/CD18 as assessed by flow cytometry. Decrements in venular shear rate induced by partial occlusion of the superior mesenteric artery in untreated animals revealed that only a minor component of L-NAME-induced leukocyte adhesion was shear rate-dependent. The L-NAME-induced adhesion was inhibited by L-arginine but not D-arginine. These data suggest that endothelium-derived NO may be an important endogenous modulator of leukocyte adherence and that impairment of NO production results in a pattern of leukocyte adhesion and emigration that is characteristic of acute inflammation.
Epstein-Barr virus latent membrane protein expression in Hodgkin and Reed-Sternberg cells.
Authors: Herbst H , Dallenbach F , Hummel M , Niedobitek G , Pileri S , Müller-Lantzsch N , Stein H
Published: May 2006

Cryostat sections from lymph nodes of 47 Hodgkin disease patients were examined by immunohistology for the Epstein-Barr virus (EBV)-encoded latent membrane protein (LMP), nuclear antigen 2, and late viral glycoprotein gp350/250. A distinct LMP-specific membrane and cytoplasmic staining was detected exclusively in Hodgkin and Reed-Sternberg cells in 18 patients (38%); EBV nuclear antigen 2 and gp350/250 immunoreactivity was absent in all instances. Thirty-two of 47 (68%) cases contained EBV-specific DNA sequences as detected by PCR, all LMP-positive cases being in this category. Our results confirm previous studies establishing the presence of EBV genomes in Hodgkin and Reed-Sternberg cells by demonstrating expression of an EBV-encoded protein in the tumor-cell population. The finding of LMP expression in the absence of EBV nuclear antigen 2 suggests a pattern of EBV gene expression different from that of B-lymphoblastoid cell lines and Burkitt lymphoma, whereas this finding shows similarities with that seen in undifferentiated nasopharyngeal carcinoma. Because the LMP gene has transforming potential, our findings support the concept of a pathoetiological role of EBV in many cases of Hodgkin disease.
Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system.
Authors: Neyfakh A A , Bidnenko V E , Chen L B
Published: May 2006

Bacillus subtilis cells selected for their resistance to rhodamine 6G demonstrated a multidrug-resistance (MDR) phenotype resembling that of mammalian MDR cells. Like MDR in mammalian cells, MDR in bacteria was mediated by the efflux of the drugs from the cells. The bacterial multidrug efflux system transported similar drugs and was sensitive to similar inhibitors as the mammalian multidrug transporter, P-glycoprotein. The gene coding for the bacterial multidrug transporter, like the P-glycoprotein gene in mammalian MDR cells, was amplified in the resistant bacteria. On the other hand, the bacterial multidrug transporter showed no sequence similarity to P-glycoprotein but exhibited an obvious homology to tetracycline efflux pumps and carbohydrate-ion symporters. These results show that the transport of structurally unrelated molecules can be mediated by members of different families of membrane transporters.
Use of a rapid, efficient inoculation method to induce papillomas by cottontail rabbit papillomavirus DNA shows that the E7 gene is required.
Authors: Brandsma J L , Yang Z H , Barthold S W , Johnson E A
Published: May 2006

A simple inoculation method to induce papillomas efficiently with cottontail rabbit papillomavirus (CRPV) DNA is described. Using a jet injector, recombinant CRPV DNA is easily delivered to 100 or more sites per rabbit and induces typical epithelial papillomas in approximately 50% of those sites. Papillomas begin to form by 3 weeks and continue to develop for up to 7 weeks, a pattern similar to that reported following infection with intact virus. This system readily lends itself to investigation of viral gene function by delivering mutant viral genomes into an immunologically intact host. Two mutations in the E7 open reading frame were introduced into the complete CRPV genome and analyzed by this method. One was a frameshift mutation encoding just nine amino-terminal amino acids of the E7 protein; the other was an in-frame insertion mutation at position 9. Both E7 mutations were in a region of homology to the 300-kDa protein binding domain of adenovirus E1A protein. Neither mutant construct was able to induce papillomas, thereby demonstrating that the E7 gene participates in this biologic function. Exploitation of this approach, which demonstrates that a papillomavirus E7 gene is involved in the induction of papillomas in vivo, should permit detailed studies into molecular mechanisms involved in papilloma induction, malignant conversion, and host immune response. The high efficiency of papilloma induction with recombinant CRPV DNA suggests that the jet injector can also be used to study the biologic effects of other genetic elements in rabbits or in other species.
Mitochondrial respiration in hummingbird flight muscles.
Authors: Suarez R K , Lighton J R , Brown G S , Mathieu-Costello O
Published: May 2006

Respiration rates of muscle mitochondria in flying hummingbirds range from 7 to 10 ml of O2 per cm3 of mitochondria per min, which is about 2 times higher than the range obtained in the locomotory muscles of mammals running at their maximum aerobic capacities (VO2max). Capillary volume density is higher in hummingbird flight muscles than in mammalian skeletal muscles. Mitochondria occupy approximately 35% of fiber volume in hummingbird flight muscles and cluster beneath the sarcolemmal membrane adjacent to capillaries to a greater extent than in mammalian muscles. Measurements of protein content, citrate synthase activity, and respiratory rates in vitro per unit mitochondrial volume reveal no significant differences between hummingbird and mammalian skeletal muscle mitochondria. However, inner membrane surface areas per unit mitochondrial volume [Sv(im,m)] are higher than those in mammalian muscle. We propose that both mitochondrial volume densities and Sv(im,m) are near their maximum theoretical limits in hummingbirds and that higher rates of mitochondrial respiration than those observed in mammals are achieved in vivo as a result of higher capacities for O2 delivery and substrate catabolism.
Influence of the cardiac myosin hinge region on contractile activity.
Authors: Margossian S S , Krueger J W , Sellers J R , Cuda G , Caulfield J B , Norton P , Slayter H S
Published: May 2006

The participation of cardiac myosin hinge in contractility was investigated by in vitro motility and ATPase assays and by measurements of sarcomere shortening. The effect on contractile activity was analyzed using an antibody directed against a 20-amino acid peptide within the hinge region of myosin. This antibody bound specifically at the hinge at a distance of 55 nm from the S1/S2 junction, was specific to human, dog, and rat cardiac myosins, did not crossreact with gizzard or skeletal myosin, and had no effect on ATPase activity of purified S1 and myofibrils. However, it completely suppressed the movement of actin filaments in in vitro motility assays and reduced active shortening of sarcomeres of skinned cardiac myocytes by half. Suppression of motion by the anti-hinge antibody may reflect a mechanical constraint imposed by the antibody upon the mobility of the S2 region of myosin. The results suggest that the steps in the mechanochemical energy transduction can be separately influenced through S2.
Correlation of histology and drug response of human tumors grown in native-state three-dimensional histoculture and in nude mice.
Authors: Vescio R A , Connors K M , Kubota T , Hoffman R M
Published: May 2006

An in vitro histoculture system in which a native-state collagen-sponge gel supports the three-dimensional growth of tumor tissue has been recently developed that allows the culture and drug response assay for most every tumor type. Important features of the histoculture system include the maintenance of three-dimensional tissue architecture and the use of histological autoradiography to measure nuclear incorporation of [3H]thymidine as an endpoint. We describe in this report in vitro-in vivo correlations for drug response and tumor histology by using human tumor xenografts grown in the native-state three-dimensional histoculture system and as xenografts in nude mice. This comparison eliminates many of the confounding variables seen in most correlative clinical trials. Results demonstrate (i) a very high preservation of in vivo tissue architecture in vitro, (ii) an 86% accuracy in vitro of predicting drug resistance in vivo using suprapharmacologic doses of drugs in vitro, and (iii) an overall predictive frequency of drug resistance and sensitivity ranging from 53% for 5-fluorouracil to 78% for doxorubicin.
Molecular mechanism of slow acetylation of drugs and carcinogens in humans.
Authors: Blum M , Demierre A , Grant D M , Heim M , Meyer U A
Published: May 2006

The acetylation polymorphism is one of the most common genetic variations in the transformation of drugs and chemicals. More than 50% of individuals in Caucasian populations are homozygous for a recessive trait and are of the "slow acetylator" phenotype. They are less efficient than "rapid acetylators" in the metabolism of numerous drugs and environmental and industrial chemicals. The acetylation polymorphism is associated with an increased risk of drug toxicity and with an increased frequency of certain cancers. We report the identification of the primary mutations in two alleles of the gene for the N-acetyltransferase (NAT; acetyl-CoA:arylamine N-acetyltransferase, EC 2.3.1.5) isozyme NAT2 associated with slow acetylation. These alleles, M1 and M2, account for more than 90% of slow acetylator alleles in the European population we have studied. M1 and M2 were identified by restriction fragment length polymorphisms with Kpn I and Msp I and subsequently cloned and sequenced. M1 and M2 each are characterized by a combination of two different point mutations, one causing an amino acid substitution (Ile-113----Thr in M1, Arg-197----Gln in M2), the other being silent (C 481----T in M1, C 282----T in M2). Functional expression of M1 and M2 and of chimeric gene constructs between mutant and wild-type NAT2 in COS-1 cells suggests that M1 causes a decrease of NAT2 protein in the liver by defective translation, whereas M2 produces an unstable enzyme. On the basis of the mutations described here and a rare mutant allele (M3) reported recently, we have developed a simple DNA amplification assay that allows the predictive genotyping of more than 95% of slow and rapid acetylator alleles and the identification of individuals at risk.
Smoking related carcinogen-DNA adducts in biopsy samples of human urinary bladder: identification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl as a major adduct.
Authors: Talaska G , al-Juburi A Z , Kadlubar F F
Published: May 2006

The prevalence of covalent modifications to DNA (carcinogen-DNA adducts) in 42 human urinary bladder biopsy samples was investigated by 32P-postlabeling methods, with enhancement by both nuclease P1 treatment and 1-butanol extraction. Total mean carcinogen-DNA adduct levels and the mean levels of several specific adducts were significantly elevated in DNA samples of 13 current smokers, as opposed to 9 never smokers or 20 ex-smokers (5 years abstinence). There was no significant difference between the latter two groups. Several DNA adducts enhanced by nuclease P1 treatment were chromatographically similar to putative hydrocarbon DNA adducts reported earlier for placenta and lung DNA samples obtained from cigarette smokers. Putative aromatic amine adducts were detected by 1-butanol extraction that were not present when the samples were treated with nuclease P1. One of these displayed chromatographic behavior identical to the predominant adduct induced by the human urinary bladder carcinogen, 4-aminobiphenyl, which is present in cigarette smoke. This adduct comigrated in several thin-layer chromatographic systems with a synthetic N-(deoxyguanosin-8-yl)-4-amino[2,2'-3H]biphenyl-3',5'-bisphosphate marker. Moreover, when this adduct was eluted from the thin-layer chromatograms of several individuals and injected onto an HPLC system, the 32P from the human bladder DNA samples coeluted in the same fraction as the tritiated synthetic N-(deoxyguanosin-8-yl)-4-aminobiphenyl marker. These data reinforce an association between cigarette smoking and DNA damage and suggest a molecular basis for the initiation of human urinary bladder cancer by cigarette smoke.
Several regions of Antennapedia and thyroid transcription factor 1 homeodomains contribute to DNA binding specificity.
Authors: Damante G , Di Lauro R
Published: May 2006

The DNA binding specificities of the homeodomains contained in thyroid transcription factor 1 and Antennapedia have been compared. The two homeodomains recognize different DNA sequences, despite the similar amino acid sequences of their recognition helix. Mutations that make the recognition helix of thyroid transcription factor 1 identical to the one contained in Antennapedia have no effects on the binding specificity of thyroid transcription factor 1. The exchange of other segments between these two homeodomains allows the identification of the regions responsible for the observed DNA binding specificities. These results indicate that amino acid residues outside of the recognition helix play an important role in the determination of the DNA binding specificities of these two homeodomains.
Interleukin 4 is important in protective immunity to a gastrointestinal nematode infection in mice.
Authors: Urban J F , Katona I M , Paul W E , Finkelman F D
Published: May 2006

Parasitic helminths typically induce components of immediate-type hypersensitivity, including elevated serum IgE, eosinophilia, and mucosal mast cells. These responses are T-cell-dependent and associated with rapid expulsion of parasitic worms from a sensitized host; existing experimental systems have failed to define the precise role of cytokines in these responses. We report here that anti-interleukin 4 or anti-interleukin 4 receptor antibodies block the polyclonal IgE response to a parasitic nematode, Heligmosomoides polygyrus, and abrogate protective immunity to the infection. In contrast, anti-interleukin 5 antibody prevented H. polygyrus-induced eosinophilia but did not prevent protection. These data provide evidence that a specific cytokine affects the physiology and survival of a parasitic nematode in the host.
Delivery of macromolecules into living cells: a method that exploits folate receptor endocytosis.
Authors: Leamon C P , Low P S
Published: May 2006

Difficulties with the nondestructive delivery of macromolecules into living cells have limited the potential applications of antibodies, genes, enzymes, peptides, and antisense oligonucleotides in biology and medicine. We have found, however, that the natural endocytosis pathway for the vitamin folate can be exploited to nondestructively introduce macromolecules into cultured cells if the macromolecule is first covalently linked to folate. Thus, treatment of KB cells with folate-conjugated ribonuclease, horseradish peroxidase, serum albumin, IgG, or ferritin allowed delivery of greater than 10(6) copies of the macromolecules within a 2-hr period. Cytochemical staining using 4-chloro-1-naphthol further demonstrated that the horseradish peroxidase retained activity for at least 6 hr after internalization. Since folate is an essential vitamin required in substantial quantities by virtually all cells, these observations may open the possibility of scientific and medical applications for many of the above macromolecules.
G alpha 16, a G protein alpha subunit specifically expressed in hematopoietic cells.
Authors: Amatruda T T , Steele D A , Slepak V Z , Simon M I
Published: May 2006

Signal-transduction pathways mediated by guanine nucleotide-binding regulatory proteins (G proteins) determine many of the responses of hematopoietic cells. A recently identified gene encoding a G protein alpha subunit, G alpha 16, is specifically expressed in human cells of the hematopoietic lineage. The G alpha 16 cDNA encodes a protein with predicted Mr of 43,500, which resembles the G q class of alpha subunits and does not include a pertussis toxin ADP-ribosylation site. In comparison with other G protein alpha subunits, the G alpha 16 predicted protein has distinctive amino acid sequences in the amino terminus, the region A guanine nucleotide-binding domain, and in the carboxyl-terminal third of the protein. Cell lines of myelomonocytic and T-cell phenotype express the G alpha 16 gene, but no expression is detectable in two B-cell lines or in nonhematopoietic cell lines. G alpha 16 gene expression is down-regulated in HL-60 cells induced to differentiate to neutrophils with dimethyl sulfoxide. Antisera generated from synthetic peptides that correspond to two regions of G alpha 16 specifically react with a protein of 42- to 43-kDa in bacterial strains that overexpress G alpha 16 and in HL-60 membranes. This protein is decreased in membranes from dimethyl sulfoxide-differentiated HL-60 cells and is not detectable in COS cell membranes. The restricted expression of this gene suggests that G alpha 16 regulates cell-type-specific signal-transduction pathways, which are not inhibited by pertussis toxin.
Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.
Authors: Barnes H J , Arlotto M P , Waterman M R
Published: May 2006

When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system.
On the role of actomyosin ATPases in regulation of ATP turnover rates during intense exercise.
Authors: Hochachka P W , Bianconcini M S , Parkhouse W S , Dobson G P
Published: May 2006

Actomyosin ATPase is the dominant ATP sink during muscle work. Its catalytic capacities in fast-twitch oxidative glycolytic fibers have long been known to exceed by about 3-fold those of slow-twitch oxidative fibers, but the relative contributions to control of metabolic rates during exercise have never been closely examined. We compared fast-twitch oxidative glycolytic and slow-twitch oxidative fibers that displayed similar mitochondrial abundance (similar activities of mitochondrial marker enzymes). During short-term, but near maximum, aerobic exercise, fast-twitch oxidative glycolytic fibers displayed ATP turnover rates that were 2-4 times higher than for slow-twitch oxidative fibers (despite similar mitochondrial metabolic capacities), implying a large ATPase contribution to control of maximum metabolic rate. Fluxes through the ATP in equilibrium ADP + Pi cycle were extremely well regulated; at the lower limit, the forward flux exceeded the backward flux by only 0.06%, whereas at the upper limit, ATPase rates exceeded ATP synthesis rates by 0.12%. This very high precision of energy coupling could not be easily explained by standard metabolic regulation models.
Enhanced hematopoietic activity of a human granulocyte/macrophage colony-stimulating factor-interleukin 3 fusion protein.
Authors: Curtis B M , Williams D E , Broxmeyer H E , Dunn J , Farrah T , Jeffery E , Clevenger W , deRoos P , Martin U , Friend D
Published: May 2006

Granulocyte/macrophage colony-stimulating factor-interleukin 3 (GM-CSF-IL-3) fusion proteins were generated by construction of a plasmid in which the coding regions of human GM-CSF and IL-3 cDNAs were connected by a synthetic linker sequence followed by subsequent expression in yeast. Both GM-CSF-IL-3 and IL-3-GM-CSF fusion proteins were purified to homogeneity and shown to bind to cell-surface receptors through either their GM-CSF or IL-3 domains. The fusion proteins exhibited enhanced receptor affinity, proliferative activity, and hematopoietic colony-stimulating activity compared with either IL-3 and/or GM-CSF alone. This suggests that GM-CSF-IL-3 fusion proteins may hold future promise as therapeutic agents.
Spermidine or spermine is essential for the aerobic growth of Saccharomyces cerevisiae.
Authors: Balasundaram D , Tabor C W , Tabor H
Published: May 2006

A null mutation in the SPE2 gene of Saccharomyces cerevisiae, encoding S-adenosylmethionine decarboxylase, results in cells with no detectable S-adenosylmethionine decarboxylase, spermidine, and spermine. This mutant has an absolute requirement for spermidine or spermine for growth; this requirement is not satisfied by putrescine. Polyamine-depleted cells show a number of microscopic abnormalities that are similar to those reported for several cell division cycle (cdc) and actin mutants. These include a striking increase in cell size, a marked decrease in budding, accumulation of vesicle-like bodies, absence of specific localization of chitin-like material, and abnormal distribution of actin-like material. The absolute requirement for polyamines for growth and the microscopic abnormalities are not seen if the cultures are grown under anaerobic conditions.
Population dynamics of natural antibodies in normal and autoimmune individuals.
Authors: Varela F , Andersson A , Dietrich G , Sundblad A , Holmberg D , Kazatchkine M , Coutinho A
Published: May 2006

We have measured the quantities of naturally occurring autoantibodies in the serum of normal, unmanipulated individuals. These changes over time following broad-band complex dynamical patterns that are similar in mouse and man. The patterns more likely reflect the network architecture of the natural antibody repertoire, regulating the activation and decay of individual clones. The temporal changes of both disease-specific and nonspecific autoantibodies are consistently modified in autoimmune individuals.