bluuebunny/crossref_metadata_2025 huggingface dataset:
Intrinsic 5-lipoxygenase activity is required for neutrophil responsivity.
Authors: Guidot D M , Repine M J , Westcott J Y , Repine J E
Published: May 2006

We found that intrinsic neutrophil 5-lipoxygenase activity was necessary for human neutrophil adherence and chemotaxis in vitro and human neutrophil-mediated acute edematous injury in isolated perfused rat lungs given interleukin 8 intratracheally. Treatment with either Zileuton (a specific reversible competitive inhibitor of 5-lipoxygenase) or MK886 (a specific irreversible inhibitor of the 5-lipoxygenase activator protein) prevented stimulated neutrophil adherence and chemotaxis (but not superoxide anion production) in vitro. Zileuton- or MK886-inhibited neutrophil chemotaxis was not restored by adding leukotriene B4 in vitro. Perfusion with neutrophils and either Zileuton or MK886, or with MK886-pretreated neutrophils (without adding MK886 to the perfusate), also prevented lung injury (reflected by lung weight gain and lung Ficoll retention) and perfusate leukotriene B4 increases in isolated rat lungs given interleukin 8 intratracheally. Again, adding leukotriene B4 to the perfusate did not damage interleukin 8-treated isolated lungs perfused with Zileuton-inhibited neutrophils. We conclude that intrinsic 5-lipoxygenase activity is required for neutrophil adherence and chemotaxis and neutrophil-mediated lung injury.
Differential expression of autoreceptors in the ascending dopamine systems of the human brain.
Authors: Meador-Woodruff J H , Damask S P , Watson S J
Published: May 2006

The tone and regulation of the brain dopaminergic projections are, in part, determined by the presence or absence of dopamine (DA) autoreceptors: rate of DA synthesis and turnover, as well as both pattern and rate of neuronal firing, are modulated by the expression and activity of these autoreceptors. The expression of dopaminergic receptors in the midbrain DA cell groups, presumably reflecting DA autoreceptors, was determined in the brains of the rat, Old World monkey, and human. In the rat, both the substantia nigra (A9) and the ventral tegmental area (A10) appear to express DA autoreceptors. In the monkey and human, however, only the projections arising from the substantia nigra express these receptors; the limbic projections originating in the ventral tegmental area lack this substrate for DA autoregulation. These results indicate that in the human, the nigrostriatal and mesocorticolimbic dopamine systems may be differentially autoregulated.
A cis-acting repressive sequence that overlaps the Rev-responsive element of human immunodeficiency virus type 1 regulates nuclear retention of env mRNAs independently of known splice signals.
Authors: Brighty D W , Rosenberg M
Published: May 2006

The Rev protein of human immunodeficiency virus type 1 (HIV-1) binds to an RNA structure, the Rev-responsive element (RRE), to enhance expression of the viral structural genes by relieving the nuclear sequestration of incompletely spliced viral transcripts. It has been suggested that nuclear retention of these mRNAs, in mammalian cells, is due to the activity of either cis-acting repressive sequence elements or to inefficient splicing signals. Expression of the HIV-1 envelope gene in transfected Drosophila cells is also dependent upon Rev coexpression and, hence, the mechanism of nuclear retention and Rev regulation are highly conserved. Here we use the Drosophila system to identify a major cis-acting repressive sequence element that overlaps the RRE and is responsible for the nuclear entrapment and Rev-dependent expression of HIV-1 env mRNAs. Moreover, the splice signals spanning env are not required for nuclear retention or Rev-dependent trans-activation of env mRNAs. We suggest that the RRE and its associated RNA structure are necessary for both the repressive and known trans-activation effects of Rev regulation.
Number, identity, and sequence of the Drosophila head segments as revealed by neural elements and their deletion patterns in mutants.
Authors: Schmidt-Ott U , González-Gaitán M , Jäckle H , Technau G M
Published: May 2006

The development of the insect head tagma involves massive rearrangements and secondary fusions of segment anlagen during embryogenesis. Due to the lack of reliable morphological markers, the number, identity, and sequence of the head segments, particularly in the pregnathal region, are still a matter of ongoing debates. We examined the complex array of internal structures of the embryonic Drosophila melanogaster head such as the sensory structures and nerves of the peripheral and stomatogastric nervous systems, and we used embryonic head mutations causing a lack of overlapping segment anlagen to unravel the segmental identity and the sequence of the neural elements. Our results provide evidence for seven distinct segments in the Drosophila head, each characterized by a specific set of sensory neurons, consistent with the proposal that insects, myriapods, and crustaceans share a monophyletic evolutionary tree from a common annelid-like ancestor.
Design of a "minimAl" homeodomain: the N-terminal arm modulates DNA binding affinity and stabilizes homeodomain structure.
Authors: Shang Z , Isaac V E , Li H , Patel L , Catron K M , Curran T , Montelione G T , Abate C
Published: May 2006

This report investigates the sequence specificity requirements for homeodomain structure and DNA binding activity by the design and synthesis of a "minimAl" homeodomain (for minimalist design and alanine scanning mutagenesis) which contains the consensus residues and in which all nonconsensus residues have been replaced with alanine. The murine homeodomain Msx served as the prototype for the minimAl homeodomain, Ala-Msx. We show that Ala-Msx binds to DNA specifically, albeit with lower affinity than Msx. A derivative of the minimAl homeodomain, Ala-Msx(NT), which contains a native rather than an alanine-substituted N-terminal arm, has similar DNA binding affinity as Msx. We show that the native N-terminal arm stabilizes the tertiary structure of the minimAl homeodomain. Although Ala-Msx resembles a molten-globule protein, the structure of Ala-Msx(NT) is similar to Msx. The requirement for an intact N-terminal arm is not unique to the minimAl homeodomain, since the N-terminal arm also promotes high-affinity binding activity and appropriate tertiary structure of Msx. Therefore, the homeodomain "scaffold" consists of consensus residues, which are sufficient for DNA recognition, and nonconsensus residues in the N-terminal arm, which are required for optimal DNA binding affinity and appropriate tertiary structure. MinimAl design provides a powerful strategy to probe homeodomain structure and function. This approach should be of general utility to study the sequence specificity requirements for structure and function of other DNA-binding domains.
Evidence for presynaptic N-methyl-D-aspartate autoreceptors in the spinal cord dorsal horn.
Authors: Liu H , Wang H , Sheng M , Jan L Y , Jan Y N , Basbaum A I
Published: May 2006

The N-methyl-D-aspartate (NMDA) receptor has been implicated in a variety of systems that undergo plastic changes in the central nervous system. We used electron microscopic immunocytochemistry with an antibody directed against an alternatively spliced exon near the C terminus of NMDAR1, the essential functional subunit of the NMDA receptor, to study the distribution of the NMDA receptor in the spinal cord and CA1 region of the hippocampus, two regions where NMDA-mediated long-term plasticity has been demonstrated. In CA1, we found that the NMDA receptor is exclusively expressed on postsynaptic structures. By contrast, in the spinal cord we found that in about one-third of labeled synapses, the receptor is located in the presynaptic terminal, immediately adjacent to the vesicle release site at the active zone. Using combined postembedding immunocytochemistry, we also showed that > 70% of the NMDA receptor immunoreactive terminals are glutamate positive, which suggests that the presynaptic NMDA receptor is an autoreceptor. Nerve ligation studies demonstrated that the receptor is transported in dorsal roots and sciatic nerve to the spinal cord and periphery, respectively. These data indicate that an NMDA autoreceptor is located in terminals of primary afferent fibers, where it could facilitate the transmission of inputs to the spinal cord by increasing the release of neurotransmitter from the primary afferent terminal.
Remodeling of cytoskeletal architecture of nonneuronal cells induced by synapsin.
Authors: Han H Q , Greengard P
Published: May 2006

The synapsins, a family of neuron-specific phosphoproteins, have been implicated in the functional and structural maturation of synapses. The cell biological basis for these effects is unknown. In vitro, the synapsins interact with cytoskeletal elements including actin. To examine, in vivo, the possible effect of the synapsins on cytoskeletal organization and cell morphology, we have transfected each of the four known members of the synapsin family into nonneuronal cells. We report here that synapsin expression in fibroblast cells gives rise to an alteration in cell morphology that is associated with formation of highly elongated processes. This morphological change is accompanied by a reorganization of filamentous actin (F-actin) characterized by disruption of existing stress fibers and formation of bundles of actin cables in the elongated processes. These results suggest that interactions of the synapsins with actin, and possibly with other cytoskeletal elements, may play a role in the morphological differentiation of neurons.
Molecular cloning and characterization of protein kinase D: a target for diacylglycerol and phorbol esters with a distinctive catalytic domain.
Authors: Valverde A M , Sinnett-Smith J , Van Lint J , Rozengurt E
Published: May 2006

A serine/threonine protein kinase that binds phorbol esters and diacylglycerol (named protein kinase D, PKD) has been identified. PKD contains membrane localization signals and a cysteine-rich repeat sequence homologous to that seen in the regulatory domain of protein kinase C (PKC). A bacterially expressed N-terminal domain of PKD exhibited high-affinity phorbol ester binding activity (Kd = 35 nM). The diacylglycerol analog 1-oleoyl-2-acetylglycerol inhibited phorbol ester binding in a dose-dependent manner. The catalytic domain of PKD contains all characteristic sequence motifs of serine protein kinases but shows only a low degree of sequence similarity to PKCs. The highest identity is with the catalytic domain of myosin light-chain kinase from Dictyostelium (41%). The bacterially expressed catalytic domain of PKD efficiently phosphorylated the exogenous peptide substrate syntide 2 in serine but did not catalyze significant phosphorylation of a variety of other substrates used by PKCs and other major second messenger regulated kinases. PKD may be an unusual component in the transduction of diacylglycerol and phorbol ester signals.
An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3.
Authors: Bett A J , Haddara W , Prevec L , Graham F L
Published: May 2006

Human adenoviruses (Ads) are attracting considerable attention because of their potential utility for gene transfer and gene therapy, for development of live viral vectored vaccines, and for protein expression in mammalian cells. Engineering Ad vectors for these applications requires a variety of reagents in the form of Ads and bacterial plasmids containing viral DNA sequences and requires different strategies for construction of vectors for different purposes. To simplify Ad vector construction and develop a procedure with maximum flexibility, efficiency, and cloning capacity, we have developed a vector system based on use of Ad5 DNA sequences cloned in bacterial plasmids. Expanded deletions in early region 1 (3180 bp) and early region 3 (2690 or 3132 bp) can be combined in a single vector that should have a capacity for inserts of up to 8.3 kb, enough to accommodate the majority of cDNAs encoding proteins with regulatory elements. Genes can be inserted into either early region 1 or 3 or both and mutations or deletions can be readily introduced elsewhere in the viral genome. To illustrate the flexibility of the system, we have introduced a wild-type early region 3 into the vectors, and to illustrate the high capacity for inserts, we have isolated a vector with two genes totaling 7.8 kb.
Murine oligodendroglial cells express nerve growth factor.
Authors: Byravan S , Foster L M , Phan T , Verity A N , Campagnoni A T
Published: May 2006

The studies reported here present evidence for the expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) by an oligodendroglial cell line and of NGF by oligodendrocytes in mouse primary culture. An immortalized oligodendroglial cell line (N19) expressing markers for immature oligodendrocytes stimulated PC12 cells to elaborate processes. Polymerase chain reaction analysis with degenerate primers indicated that the N19 cells expressed the mRNAs for the neurotrophic factors NGF and BDNF. Northern blot analysis confirmed that the N19 cells expressed the 1.3-kb NGF mRNA and the 1.4- and 4-kb BDNF mRNAs. In situ hybridization histochemistry identified the presence of NGF mRNAs in 9-day primary oligodendroglial cultures. Combined immunocytochemistry and in situ hybridization histochemistry colocalized NGF mRNA within primary cultured cells that immunostained for the oligodendrocyte marker galactocerebroside (GC). Double-immunofluorescence analysis also colocalized NGF protein within GC+ cells and within A2B5+ cells, a marker for oligodendrocyte progenitors. These results show that oligodendroglia and their precursor cells can express the neurotrophic factor NGF. They suggest that cells in the oligodendrocyte lineage may play an active role in neurite extension through fiber tracts in addition to myelination.
Cellular signaling by an agonist-activated receptor/Gs alpha fusion protein.
Authors: Bertin B , Freissmuth M , Jockers R , Strosberg A D , Marullo S
Published: May 2006

The consequences of agonist-dependent activation of guanine nucleotide-binding protein (G protein)-coupled receptors vary from cell to cell, depending on a complex network of regulations between components of the signaling cascade. Specific interactions between receptors, G proteins, and effectors are difficult to analyze in intact cells. Engineering of receptor-transducer fusion proteins might be an effective strategy to target cellular effectors more efficiently and specifically. As a model, we evaluated the ability of a fusion protein of beta 2-adrenergic receptor bound to the alpha subunit of adenylyl cyclase-stimulatory G protein (Gs alpha) to restore the defective activation of adenylyl cyclase in S49 cyc- cells that lack endogenous Gs alpha. The coupling between the two partners of the fusion protein was functional, and the agonist-dependent activation of the effector was more potent and more productive in transfected than in wild-type S49 cells. The covalent link between receptor and Gs alpha could thus convey an advantage over freely interacting components. Such receptor-G alpha fusion proteins may help to elucidate the complex interactions between members of signaling pathways and may also constitute a useful tool for studying the effects of single effector activation.
Monoclonal antibody-superantigen fusion proteins: tumor-specific agents for T-cell-based tumor therapy.
Authors: Dohlsten M , Abrahmsén L , Björk P , Lando P A , Hedlund G , Forsberg G , Brodin T , Gascoigne N R , Förberg C , Lind P
Published: May 2006

The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on major histocompatibility complex (MHC) class II molecules. To develop a tumor-specific superantigen for cancer therapy, we have made a recombinant fusion protein of SEA and the Fab region of the C215 monoclonal antibody specific for human colon carcinoma cells. SEA as part of a fusion protein showed a > 10-fold reduction in MHC class II binding compared to native SEA, and accordingly, the affinity of the FabC215-SEA fusion protein for the C215 tumor antigen was approximately 100-fold stronger than to MHC class II molecules. The FabC215-SEA fusion protein efficiently targeted T cells to lyse C215+ MHC class II- human colon carcinoma cells, which demonstrates functional substitution of the MHC class II-dependent presentation of SEA with tumor specificity. Treatment of mice carrying B16 melanoma cells expressing a transfected C215 antigen resulted in 85-99% inhibition of tumor growth and allowed long-term survival of animals. The therapeutic effect was dependent on antigen-specific targeting of the FabC215-SEA fusion protein, since native SEA and an antigen-irrelevant FabC242-SEA fusion protein did not influence tumor growth. The results suggest that Fab-SEA fusion proteins convey superantigenicity on tumor cells, which evokes T cells to suppress tumor growth.
Transgenic engineering of neuromuscular junctions in Xenopus laevis embryos transiently overexpressing key cholinergic proteins.
Authors: Shapira M , Seidman S , Sternfeld M , Timberg R , Kaufer D , Patrick J , Soreq H
Published: May 2006

To examine the role of key cholinergic proteins in the formation of neuromuscular junctions (NMJs), we expressed DNAs encoding the mouse muscle nicotinic acetylcholine receptor (nAChR) or human brain and muscle acetylcholinesterase (hAChE) in developing Xenopus laevis embryos. Acetylthiocholine hydrolysis and alpha-bungarotoxin binding in homogenates of transgenic embryos revealed transient overexpression of the respective proteins for at least 4 days postfertilization. Moreover, hAChE injection induced an approximately 2-fold increase in endogenous Xenopus nAChR. Electron microscopy coupled with cytochemical staining for AChE activity revealed that AChE-stained areas, which reached 0.17 microns2 in NMJs of control embryos raised at 21 degrees C, increased up to 0.53 and 0.60 microns2 in nAChR and hAChE transgenics, respectively. These increases coincided with the appearance of a class of large NMJs with average postsynaptic lengths up to 1.8-fold greater than controls. As much as 57% and 34% of the NMJs in animals transgenic for nAChR and hAChE, respectively, displayed AChE activity in nerve terminals in addition to muscle labeling, as compared with 10% nerve-labeled NMJs in control animals. Moreover, area, but not length values, were > 2-fold larger in hAChE-expressing NMJs labeled in their nerve terminals than in those labeled in muscle alone, reflecting a hAChE-induced increase in synaptic cleft width. These findings indicate that modulation of cholinergic neurotransmission in NMJs modifies the features of nerve-muscle connections.
Creatine kinase (CK) in skeletal muscle energy metabolism: a study of mouse mutants with graded reduction in muscle CK expression.
Authors: van Deursen J , Ruitenbeek W , Heerschap A , Jap P , ter Laak H , Wieringa B
Published: May 2006

To understand better the role of the creatine kinase (CK)/phosphocreatine system in muscle bioenergetics, a series of mouse mutants with subnormal muscle CK (M-CK) expression has been generated. Here we compare the phenotypes of mice deficient in M-CK (M-CK-/-) and M-CK leaky-mutant mice, which carry a targeted insertion of a hygromycin B-poly(A) resistance cassette in the second M-CK intron. Mice homozygous for this M-CK allele (M-CKI/I) have a 3-fold reduction of dimeric muscle CK enzyme activity, whereas compound heterozygotes with the null M-CK allele (M-CKI/-) display a 6-fold reduction. Unlike M-CK-/- mice, these mutants have no increased glycogen content or glycogen consumption in their fast fibers. The intermyofibrillar mitochondrial volume of these fibers is also normal, suggesting that energy transport via the CK/phosphocreatine system may function at low myofibrillar M-band CK levels. Conversely, the flux of energy through the CK reaction is still not visible by means of 31P NMR spectroscopy, indicating that relatively high levels of M-CK expression (> 34% of normal) are required to generate CK fluxes detectable by this technique. The ability of muscles to perform burst activity is also subnormal and closely correlates with the level of M-CK expression.
The pleckstrin homology domain of Bruton tyrosine kinase interacts with protein kinase C.
Authors: Yao L , Kawakami Y , Kawakami T
Published: May 2006

Bruton tyrosine kinase (EC 2.7.1.112) [Btk, encoded by Btk in mice and BTK in humans (formerly known as atk, BPK, or emb)], which is variously mutated in chromosome X-linked agammaglobulinemia patients and X-linked immunodeficient (xid) mice, has the pleckstrin homology (PH) domain at its amino terminus. The PH domain of Btk expressed as a bacterial fusion protein directly interacts with protein kinase C in mast cell lysates. Evidence was obtained that Btk is physically associated with protein kinase C in intact murine mast cells as well. Both Ca(2+)-dependent (alpha, beta I, and beta II) and Ca(2+)-independent protein kinase C isoforms (epsilon and zeta) in mast cells interact with the PH domain of Btk in vitro, and protein kinase C beta I is associated with Btk in vivo. Btk served as a substrate of protein kinase C, and its enzymatic activity was down-regulated by protein kinase C-mediated phosphorylation. Furthermore, depletion or inhibition of protein kinase C with pharmacological agents resulted in an enhancement of the tyrosine phosphorylation of Btk induced by mast cell activation.
Differential expression of two fibroblast growth factor-receptor genes is associated with malignant progression in human astrocytomas.
Authors: Yamaguchi F , Saya H , Bruner J M , Morrison R S
Published: May 2006

Malignant astrocytomas, which are highly invasive, vascular neoplasms, compose the majority of nervous system tumors in humans. Elevated expression of fibroblast growth factors (FGFs) in astrocytomas has implicated the FGF family of mitogens in the initiation and progression of astrocyte-derived tumors. In this study, we demonstrated that human astrocytomas undergo parallel changes in FGF-receptor (FGFR) expression during their progression from a benign to a malignant phenotype. FGFR type 2 (BEK) expression was abundant in normal white matter and in all low-grade astrocytomas but was not seen in malignant astrocytomas. Conversely, FGFR type 1 (FLG) expression was absent or barely detectable in normal white matter but was significantly elevated in malignant astrocytomas. Malignant astrocytomas also expressed an alternatively spliced form of FGFR-1 (FGFR-1 beta) containing two immunoglobulin-like disulfide loops, whereas normal human adult and fetal brains expressed a receptor form (FGFR-1 alpha) containing three immunoglobulin-like disulfide loops. Intermediate grades of astrocytic tumors exhibited a gradual loss of FGFR-2 and a shift in expression from FGFR-1 alpha to FGFR-1 beta as they progressed from benign to malignant phenotype. These results suggest that differential expression and alternative splicing of FGFRs may be critical in the malignant progression of astrocytic tumors.
The polymorphic subtelomeric regions of Plasmodium falciparum chromosomes contain arrays of repetitive sequence elements.
Authors: de Bruin D , Lanzer M , Ravetch J V
Published: May 2006

The human malaria parasite Plasmodium falciparum exhibits a high degree of chromosomal polymorphism, which may contribute to its ability to evade host defenses. The analysis of parasite chromosomes has revealed that these polymorphisms are confined to the subtelomeric regions, which are transcriptionally silent and contain repetitive sequence elements. Several subtelomeric repetitive elements have been isolated and mapped by using P. falciparum yeast artificial chromosome (YAC) clones. Structural analysis of parasite telomeric and subtelomeric YAC clones demonstrated that these repetitive elements are conserved between P. falciparum chromosome ends. We suggest that these subtelomeric elements promote chromosome pairing in P. falciparum and facilitate meiotic recombination and gene conversion between telomere-proximal genes.
Function of a human cyclin gene as an oncogene.
Authors: Hinds P W , Dowdy S F , Eaton E N , Arnold A , Weinberg R A
Published: May 2006

The cyclin D1 (PRAD1, CCND1) gene is affected by translocations and amplification in the genomes of a number of human tumors, suggesting that these changes confer growth advantage on developing tumor cell clones. We show here that in cultured cells, a cDNA clone of the cyclin D1 gene can contribute to cell transformation by complementing a defective adenovirus E1A oncogene. In such cells, this candidate oncogene indeed functions like an oncogene, suggesting a similar role in tumor progression in vivo.
Allelic associations of two polymorphic microsatellites in intron 40 of the human von Willebrand factor gene.
Authors: Pena S D , de Souza K T , de Andrade M , Chakraborty R
Published: May 2006

At intron 40 of the von Willebrand factor (vWF) gene, two GATA-repeat polymorphic sites exist that are physically separated by 212 bp. At the first site (vWF1 locus), seven segregating repeat alleles were observed in a Brazilian Caucasian population, and at the second (vWF2 locus) there were eight alleles, detected through PCR amplifications of this DNA region. Haplotype analysis of individuals revealed 36 different haplotypes in a sample of 338 chromosomes examined. Allele frequencies between generations and gender at each locus were not significantly different, and the genotype frequencies were consistent with their Hardy-Weinberg expectations. Linkage disequilibrium between loci is highly significant with positive allele size association; that is, large alleles at the loci tend to occur together, and so do the small alleles. Variability at each locus appeared to have arisen in a stepwise fashion, suggesting replication slippage as a possible mechanism of production of new alleles. However, we observed an increased number of haplotypes, in contrast with the predictions of a stepwise production of variation in the entire region, suggesting some form of "cooperative" changes between loci that could be due to either gene conversion, or a common control mechanism of production of new variation at these repeat polymorphism sites. The high degree of polymorphism (gene diversity values of 72% and 78% at vWF1 and vWF2, respectively, and of 93% at the haplotype level) makes these markers informative for paternity testing, genetic counseling, and individual-identification purposes.
Eye position and eye velocity integrators reside in separate brainstem nuclei.
Authors: Pastor A M , De la Cruz R R , Baker R
Published: May 2006

Two types of central nervous system integrators are critical for oculomotor performance. The first integrates velocity commands to create position signals that hold fixation of the eye. The second stores relative velocity of the head and visual surround to stabilize gaze both during and after the occurrence of continuous self and world motion. We have used recordings from single neurons to establish that the "position" and "velocity" integrators for horizontal eye movement occupy adjacent, but nonoverlapping, locations in the goldfish medulla. Lidocaine inactivation of each integrator results in the eye movement deficits expected if horizontal eye position and velocity signals are processed separately. These observations also indicate that each brainstem compartment generates and stores these signals. Consequently, each integrator exhibits functional autonomy. Therefore, we propose that the intrinsic electrophysiological properties of the constituent neurons in each brainstem subnucleus may be sufficient for producing integrator rhythmicity.
Adaptation in brain glucose uptake following recurrent hypoglycemia.
Authors: Boyle P J , Nagy R J , O'Connor A M , Kempers S F , Yeo R A , Qualls C
Published: May 2006

Brain glucose metabolism is impaired during hypoglycemia, but, if sustained, brain metabolism reverts to normal in animal models--data in man are lacking. We tested the hypothesis that adaptations occur to allow maintenance of normal rates of brain glucose uptake (BGU) following recurrent hypoglycemia in man. Twelve normal humans were studied over 4 days. On the initial day, arterial plasma glucose concentrations were decreased from 4.72 to 2.50 mmol/liter in five 0.56 mmol/liter steps. Cerebral blood flow, brain arteriovenous glucose difference, BGU, and cognitive function were quantitated at each step. BGU was initially impaired at the 3.61 mmol/liter glucose step (P = 0.04) and was antedated by increments in epinephrine that began at 4.16 mmol/liter (P = 0.03). The onset of hypoglycemic symptoms occurred during the 3.61 mmol/liter glucose step (P = 0.02), whereas tests of cognitive function generally deteriorated at the 3.05 mmol/liter step (P < 0.05). During the next 56 hr, mean glucose concentrations were kept at 2.9 +/- 0.1 mmol/liter and reached normal only during meals. The stepped clamp protocol was repeated beginning at 4.16 mmol/liter on the last day. No decrement in BGU was observed at any step; cognitive function was preserved until significantly lower glucose concentrations on the final day relative to the first (P = 0.04). Subjects remained asymptomatic of hypoglycemia until they reached a glucose concentration of 2.50 mmol/liter (P < 0.001 vs. day 1), while initial increments in all counterregulatory hormones were forestalled to lower glucose steps than on day 1. Therefore, adaptations occur that allow normal BGU and cerebral function to be maintained during recurrent systemic hypoglycemia. Counterregulatory events that should result in symptoms of hypoglycemia and increments in endogenous glucose production are prevented until extremely subnormal glucose concentrations.
Evolutionary relationships of lactate dehydrogenases (LDHs) from mammals, birds, an amphibian, fish, barley, and bacteria: LDH cDNA sequences from Xenopus, pig, and rat.
Authors: Tsuji S , Qureshi M A , Hou E W , Fitch W M , Li S S
Published: May 2006

The nucleotide sequences of the cDNAs encoding LDH (EC 1.1.1.27) subunits LDH-A (muscle), LDH-B (liver), and LDH-C (oocyte) from Xenopus laevis, LDH-A (muscle) and LDH-B (heart) from pig, and LDH-B (heart) and LDH-C (testis) from rat were determined. These seven newly deduced amino acid sequences and 22 other published LDH sequences, and three unpublished fish LDH-A sequences kindly provided by G. N. Somero and D. A. Powers, were used to construct the most parsimonious phylogenetic tree of these 32 LDH subunits from mammals, birds, an amphibian, fish, barley, and bacteria. There have been at least six LDH gene duplications among the vertebrates. The Xenopus LDH-A, LDH-B, and LDH-C subunits are most closely related to each other and then are more closely related to vertebrate LDH-B than LDH-A. Three fish LDH-As, as well as a single LDH of lamprey, also seem to be more related to vertebrate LDH-B than to land vertebrate LDH-A. The mammalian LDH-C (testis) subunit appears to have diverged very early, prior to the divergence of vertebrate LDH-A and LDH-B subunits, as reported previously.
Advanced glycation end products up-regulate gene expression found in diabetic glomerular disease.
Authors: Yang C W , Vlassara H , Peten E P , He C J , Striker G E , Striker L J
Published: May 2006

Several lines of evidence suggest that the excessive accumulation of extracellular matrix in the glomeruli of diabetic kidneys may be due to reactive intermediates forming between glucose and matrix proteins called advanced glycation end products (AGEs). Normal mice received AGE-modified mouse serum albumin i.p. for 4 weeks, and glomerular extracellular matrix, growth factor mRNA levels, and morphology were examined. We found that AGE induced an increase in glomerular extracellular matrix alpha 1(IV) collagen, laminin B1, and transforming growth factor beta 1 mRNA levels, as measured by competitive PCR, as well as glomerular hypertrophy. The AGE response was specific because the coadministration of an AGE inhibitor, aminoguanidine, reduced all these changes. We conclude that AGEs affected expression of genes implicated in diabetic kidney disease and may play a major role in nephropathy.
Prevention of immunotoxin-mediated vascular leak syndrome in rats with retention of antitumor activity.
Authors: Siegall C B , Liggitt D , Chace D , Tepper M A , Fell H P
Published: May 2006

Immunotoxins are hybrid molecules composed of a cell-surface binding domain and a protein toxin moiety that together target specific cell populations for elimination. These agents represent a promising approach for the treatment of many human diseases, most notably cancer. However, it has recently become clear that many immunotoxins when used in human clinical trials induce vascular leak syndrome (VLS), restricting the administration of doses necessary to achieve good therapeutic responses. The lack of an appropriate animal model has hindered efforts to understand and prevent immunotoxin-induced VLS. We have found that in rats, intravenous administration of the single-chain immunotoxin BR96 sFv-PE40 results in symptoms that closely resemble VLS seen in human immunotoxin trials. A large fluid accumulation in the thoracic cavity was observed, along with an increase in hematocrit and body weight and a decrease in serum albumin. The VLS was apparent within 24 hr after administration of immunotoxin and was seen in both immunocompetent and athymic rats. Similar symptoms were not found in mice even at lethal doses. Prophylactic administration of the corticosteroid dexamethasone resulted in prevention of VLS and survival of rats injected with what would otherwise be lethal doses of BR96 sFv-PE40. Prophylactic treatment with dexamethasone in rats xenografted with human tumors either did not inhibit or minimally inhibited the antitumor activity of BR96 sFv-PE40. The use of prophylactic corticosteroids should be considered for immunotoxin clinical trials, since it may improve therapeutic efficacy by decreasing the dose-limiting toxicity of VLS.
Coupling of RNA displacement and intrinsic termination in transcription from synthetic RNA.DNA bubble duplex constructs.
Authors: Daube S S , Hart C R , von Hippel P H
Published: May 2006

Functional transcription elongation complexes can be formed by adding RNA polymerase in trans to a preformed nucleic acid construct. This construct consists of a double-stranded DNA fragment that contains a noncomplementary (permanent DNA bubble) region into which an RNA primer oligonucleotide has been hybridized. By ligating a DNA fragment containing the strong intrinsic terminator T7Te to the RNA.DNA bubble duplex, we show here that Escherichia coli core RNA polymerase-catalyzed transcription, initiated from such a construct, terminates at the predicted position. Furthermore, we show that the termination efficiency obtained is comparable to that observed in a control reaction initiated with the E. coli holopolymerase from the T7A1 promoter if an RNA oligomer trap is used to permit proper displacement of the nascent RNA from the DNA template strand. The trap oligomer is complementary to the template strand of the permanent DNA bubble and prevents rehybridization of the nascent RNA at this site. Varying the amount of RNA trap that is added permits us to modulate the extent of total RNA displacement. Our results show that RNA displacement and termination efficiency are directly correlated, suggesting that intrinsic termination requires that the nascent RNA be free to assume its solution conformation. Several models of intrinsic termination are presented and discussed in light of these data.
A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes.
Authors: Yee J K , Miyanohara A , LaPorte P , Bouic K , Burns J C , Friedmann T
Published: May 2006

Retroviral vectors have been central components in many studies leading to human gene therapy. However, the generally low titers and inefficient infectivity of retroviral vectors in human cells have limited their use. We previously reported that the G protein of vesicular stomatitis virus can serve as the exclusive envelope protein component for one specific retroviral vector, LGRNL, that expresses vesicular stomatitis virus G. We now report a more useful general transient transfection scheme for producing very high-titer vesicular stomatitis virus G-enveloped pseudotypes from any Moloney murine leukemia-based retroviral vector without having to rely on the expression of the cytotoxic G protein from the retroviral vector itself. We also demonstrate very high efficiency of infection with a pseudotyped lacZ vector in primary mouse hepatocytes. We suggest that pseudotyped retroviral vectors carrying reporter genes will permit genetic studies in many previously inaccessible vertebrate and invertebrate systems. Furthermore, because these vectors represent retroviral vectors of sufficiently high titer to allow efficient direct retroviral-mediated in vivo gene transfer, we also suggest that pseudotyped vectors carrying potentially therapeutic genes will become useful to test the potential for in vivo gene therapy.
E1 protein of human papillomavirus type 1a is sufficient for initiation of viral DNA replication.
Authors: Gopalakrishnan V , Khan S A
Published: May 2006

Previous studies on transient replication of papillomaviruses have shown an absolute requirement for the viral E1 and E2 proteins in DNA replication. Here we demonstrate that for human papillomavirus type 1a (HPV-1a) DNA, the E1 protein alone is sufficient for in vivo replication of plasmids containing the viral origin of replication. Replication was origin-specific and required the presence of a DNA sequence containing a putative E1 binding site, but the E2 binding sites were dispensable. In the presence of the E1 protein, E2 stimulated replication of plasmids containing the E1 and E2 binding sites, but no stimulation was observed when the origin plasmids lacked E2 binding sites. Conversely, in the presence of E1 alone, the E2 binding sites did not affect replication. Plasmids containing the replication origins of HPV-6b, HPV-18, and bovine papillomavirus type 1 (BPV-1) also replicated efficiently in the presence of the HPV-1a E1 and E2 proteins. However, plasmids containing the origins of HPV-6b and HPV-18 failed to replicate in the presence of HPV-1a E1 alone, whereas a plasmid containing the BPV-1 origin replicated to lower levels than the HPV-1a origin-containing plasmid. These results suggest that replication from papillomaviral origins in the presence of E1 alone is presumably dependent on the strength of E1-origin interactions. Additionally, E1-dependent replication is stimulated by the E2 protein in the presence of E2 binding sites.
Functional magnetic resonance studies of the reorganization of the human hand sensorimotor area after unilateral brain injury in the perinatal period.
Authors: Cao Y , Vikingstad E M , Huttenlocher P R , Towle V L , Levin D N
Published: May 2006

Functional magnetic resonance imaging was used to map the hand sensorimotor area of hemiparetic adolescents and young adults who had suffered unilateral brain damage in the perinatal period. Unlike normal subjects, who exhibit cortical activation primarily contralateral to voluntary finger movements, the hemiparetic patients' intact hemispheres were equally activated by contralateral and ipsilateral finger movements. Our findings are consistent with previous clinical observations and animal experiments which suggest that the immature brain is able to reorganize in response to focal injury.
Reversible unfolding of fibronectin type III and immunoglobulin domains provides the structural basis for stretch and elasticity of titin and fibronectin.
Authors: Erickson H P
Published: May 2006

The elastic protein titin comprises a tandem array of fibronectin type III and immunoglobulin domains, which are structurally similar 7-strand beta-sandwiches. A proposed mechanism for stretching titin, by sequential denaturation of individual fibronectin type III-immunoglobulin domains in response to applied tension, is analyzed here quantitatively. The folded domain is approximately 4 nm long, and the unraveled polypeptide can extend to 29 nm, providing a 7-fold stretch over the relaxed length. Elastic recoil is achieved by refolding of the denatured domains when the force is released. The critical force required to denature a domain is calculated to be 3.5-5 pN, based on a net free energy for denaturation of 7-14 kcal/mol, plus 5 kcal/mol to extend the polypeptide (1 cal = 4.184 J). This force is comparable to the 2- to 7-pN force generated by single myosin or kinesin molecules. The force needed to pull apart a noncovalent protein-protein interface is estimated here to be 10-30 pN, implying that titin will stretch internally before the molecule is pulled from its attachment at the Z band. Many extracellular matrix and cell adhesion molecules, such as fibronectin, contain tandem arrays of fibronectin type III domains. Both single molecules and matrix fibers should have elastic properties similar to titin.
Complement C3 participation in monocyte adhesion to different surfaces.
Authors: McNally A K , Anderson J M
Published: May 2006

As part of an ongoing investigation into the role of the monocyte/macrophage in biocompatibility, a major goal is to identify the adhesion mechanisms that initiate and promote the observed in vivo morphologic progression of monocyte-to-macrophage-to-foreign body giant cell on biomaterials. We have exploited differently modified polystyrenes, specific component-depleted sera, and monoclonal antibodies (mAbs) to leukocyte integrins to ask what adhesion mechanisms mediate human blood monocyte adhesion to different surfaces in vitro. Preliminary findings are that monocyte interactions with fluorinated, siliconized, nitrogenated, and oxygenated surfaces are reduced by 50-100% when complement component C3-depleted serum is used for adsorption; reductions vary with material surface properties. Adhesion is restored on all surfaces when C3-depleted serum is replenished with purified C3. Monocyte adhesion to serum-adsorbed surfaces is inhibited by mAbs to the leukocyte integrin beta subunit, CD18 (mAbs 60.3 and MHM23), and partially inhibited by a mAb to the alpha subunit, CD11b (mAb 60.1), suggesting adhesive interactions between adsorbed C3bi (the hemolytically inactive form of the C3b fragment) and the leukocyte integrin CD11b/CD18. However, adsorbed fibrinogen reduces the effectiveness of these mAbs, indicating that alternative adhesion mechanisms may operate depending on the propensities of critical adhesion-mediating components to be adsorbed onto different surfaces.
Imaging nuclear pores of aldosterone-sensitive kidney cells by atomic force microscopy.
Authors: Oberleithner H , Brinckmann E , Schwab A , Krohne G
Published: May 2006

In nuclei of renal target cells, aldosterone enhances transcriptional activity followed by the translocation of specific RNA molecules across the nuclear envelope. Trafficking between cell nucleus and cytoplasm occurs via nuclear pore complexes (NPCs) located in the double-layered nuclear envelope. We investigated the nucleocytoplasmic transport route by structure-function analysis at subcellular level in quiescent and aldosterone-stimulated cells. With atomic-force microscopy (AFM) we imaged individual pores of the nuclear surface of cultured kidney cells and related the number of pores per micron2 to nuclear envelope conductance (Gn, per micron2) evaluated electrically by current injection into the isolated nucleus. NPCs were equally distributed resembling "donut-like" structures with outer diameters of 134 +/- 12 nm (n = 50), each equipped with a central channel. Six hours of aldosterone exposure (0.1 microM) increased the number of NPCs per micron 2 of nuclear surface from 7.4 +/- 0.4 to 9.8 +/- 0.4 (n = 12; P < 0.01). At the same time Gn rose from 6900 +/- 520 to 9600 +/- 610 pS/micron2 paralleled by an increase of the intranuclear electrical potential from -2.8 +/- 0.2 to -6.2 +/- 0.4 mV (n = 18; P < 0.01). Assuming that NPCs represent the sole conductive pathway in the nuclear envelope, we calculate a mean single NPC conductance of 932 and 980 pS, in the absence and presence of aldosterone, respectively. We conclude that aldosterone facilitates nucleocytoplasmic transport by increasing the number of NPCs but not by modifying their biophysical properties. Possibly, aldosterone controls similar transport mechanisms in both plasma membrane and nuclear envelope.
Molecular cloning of a gene encoding an arabinogalactan-protein from pear (Pyrus communis) cell suspension culture.
Authors: Chen C G , Pu Z Y , Moritz R L , Simpson R J , Bacic A , Clarke A E , Mau S L
Published: May 2006

Arabinogalactan-proteins (AGPs) are proteoglycans containing a high proportion of carbohydrate (typically > 90%) linked to a protein backbone rich in hydroxyproline (Hyp), Ala, Ser, and Thr. They are widely distributed in plants and may play a role in development. The structure of the carbohydrate of some AGPs is known in detail but information regarding the protein backbone is restricted to a few peptide sequences. Here we report isolation and partial amino acid sequencing of the protein backbone of an AGP. This AGP is a member of one of four major groups of AGPs isolated from the filtrate of pear cell suspension culture. A cDNA encoding this protein backbone (145 amino acids) was cloned; the deduced protein is rich in Hyp, Ala, Ser, and Thr, which together account for > 75% of total residues. It has three domains, an N-terminal secretion signal, a central hydrophilic domain containing all of the Pro residues, and a hydrophobic C-terminal domain that is predicted to be a transmembrane helix. Approximately 93% of the Pro residues are hydroxylated and hence are potential sites for glycosylation.
Mitochondrial carbonic anhydrase (isozyme V) in mouse and rat: cDNA cloning, expression, subcellular localization, processing, and tissue distribution.
Authors: Nagao Y , Srinivasan M , Platero J S , Svendrowski M , Waheed A , Sly W S
Published: May 2006

When the human cDNA, isolated on the basis of homology to the murine carbonic anhydrase (CA) "Y" was expressed in COS cells, the human CA was targeted to and processed in mitochondria, as expected for CA-V. However, tissue distribution reported for the corresponding mouse CA Y mRNA was much more limited than that reported for the distribution of CA-V immunostaining in rat tissues. To determine whether the murine cDNA actually encodes a mitochondrial CA activity and to compare the tissue distribution of the homologous murine and rat gene products, we used reverse transcription-PCR to reisolate the murine CA-V candidate cDNA and used the murine cDNA probe to isolate the homologous rat cDNA. We compared the two cDNA sequences, the activities they expressed after transfection of COS cells, and the sites of N-terminal processing of expressed products. In addition, we used antibodies to the C-terminal peptides predicted from each cDNA to compare distribution of CA-V in mouse and rat tissues and to identify CA-Vs in mitochondria isolated from mouse and rat liver. From these studies, we conclude that both mouse and rat CA-V candidate cDNAs encode active CAs that are targeted to and processed in mitochondria and that there are real differences in tissue distribution of CA-V between mouse and rat. However, the findings that are M(r) of CA-V in rat tissues is smaller than that previously reported and that the tissue distribution also differs lead us to conclude that the antibody used in prior reports most likely misidentified another antigen in rat tissues as CA-V.