bluuebunny/crossref_metadata_2025 huggingface dataset:
Directing gene expression to cerebellar granule cells using γ-aminobutyric acid type A receptor
α6
subunit transgenes
Authors: Bahn Sabine , Jones Alison , Wisden William
Published: July 2002

Expression of the γ-aminobutyric acid type A receptor
α6
subunit gene is restricted to differentiated granule cells of the cerebellum and cochlear nucleus. The mechanisms underlying this limited expression are unknown. Here we have characterized the expression of a series of
α6
-based transgenes in adult mouse brain. A DNA fragment containing a 1-kb portion upstream of the start site(s), together with exons 1–8, can direct high-level cerebellar granule cell-specific reporter gene expression. Thus powerful granule cell-specific determinants reside within the 5′ half of the
α6
subunit gene body. This intron-containing transgene appears to lack the cochlear nucleus regulatory elements. It therefore provides a cassette to deliver gene products solely to adult cerebellar granule cells.
Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector
Authors: Miyoshi Hiroyuki , Takahashi Masayo , Gage Fred H. , Verma Inder M.
Published: July 2002

The development of methods for efficient gene transfer to terminally differentiated retinal cells is important to study the function of the retina as well as for gene therapy of retinal diseases. We have developed a lentiviral vector system based on the HIV that can transduce terminally differentiated neurons of the brain
in vivo
. In this study, we have evaluated the ability of HIV vectors to transfer genes into retinal cells. An HIV vector containing a gene encoding the green fluorescent protein (GFP) was injected into the subretinal space of rat eyes. The GFP gene under the control of the cytomegalovirus promoter was efficiently expressed in both photoreceptor cells and retinal pigment epithelium. However, the use of the rhodopsin promoter resulted in expression predominantly in photoreceptor cells. Most successfully transduced eyes showed that photoreceptor cells in >80% of the area of whole retina expressed the GFP. The GFP expression persisted for at least 12 weeks with no apparent decrease. The efficient gene transfer into photoreceptor cells by HIV vectors will be useful for gene therapy of retinal diseases such as retinitis pigmentosa.
Reversible antisense inhibition of Shaker-like Kv1.1 potassium channel expression impairs associative memory in mouse and rat
Authors: Meiri Noam , Ghelardini Carla , Tesco Giuseppina , Galeotti Nicoletta , Dahl Dennis , Tomsic Daniel , Cavallaro Sebastiano , Quattrone Alessandro , Capaccioli Sergio , Bartolini Alessandro , Alkon Daniel L.
Published: July 2002

Long-term memory is thought to be subserved by functional remodeling of neuronal circuits. Changes in the weights of existing synapses in networks might depend on voltage-gated potassium currents. We therefore studied the physiological role of potassium channels in memory, concentrating on the Shaker-like Kv1.1, a late rectifying potassium channel that is highly localized within dendrites of hippocampal CA3 pyramidal and dentate gyrus granular cells. Repeated intracerebroventricular injection of antisense oligodeoxyribonucleotide to Kv1.1 reduces expression of its particular intracellular mRNA target, decreases late rectifying K
+
current(s) in dentate granule cells, and impairs memory but not other motor or sensory behaviors, in two different learning paradigms, mouse passive avoidance and rat spatial memory. The latter, hippocampal-dependent memory loss occurred in the absence of long-term potentiation changes recorded both from the dentate gyrus or CA1. The specificity of the reversible antisense targeting of mRNA in adult animal brains may avoid irreversible developmental and genetic background effects that accompany transgenic “knockouts”.
Expression of a β-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice
Authors: Rockman Howard A. , Chien Kenneth R. , Choi Dong-Ju , Iaccarino Guido , Hunter John J. , Ross John , Lefkowitz Robert J. , Koch Walter J.
Published: July 2002

Heart failure is accompanied by severely impaired β-adrenergic receptor (βAR) function, which includes loss of βAR density and functional uncoupling of remaining receptors. An important mechanism for the rapid desensitization of βAR function is agonist-stimulated receptor phosphorylation by the βAR kinase (βARK1), an enzyme known to be elevated in failing human heart tissue. To investigate whether alterations in βAR function contribute to the development of myocardial failure, transgenic mice with cardiac-restricted overexpression of either a peptide inhibitor of βARK1 or the β
2
AR were mated into a genetic model of murine heart failure (
MLP
−/−
).
In vivo
cardiac function was assessed by echocardiography and cardiac catheterization. Both
MLP
−/−
and
MLP
−/−
/β
2
AR mice had enlarged left ventricular (LV) chambers with significantly reduced fractional shortening and mean velocity of circumferential fiber shortening. In contrast,
MLP
−/−
/βARKct mice had normal LV chamber size and function. Basal LV contractility in the
MLP
−/−
/βARKct mice, as measured by LV dP/dtmax, was increased significantly compared with the
MLP
−/−
mice but less than controls. Importantly, heightened βAR desensitization in the
MLP
−/−
mice, measured
in vivo
(responsiveness to isoproterenol) and
in vitro
(isoproterenol-stimulated membrane adenylyl cyclase activity), was completely reversed with overexpression of the βARK1 inhibitor. We report here the striking finding that overexpression of this inhibitor prevents the development of cardiomyopathy in this murine model of heart failure. These findings implicate abnormal βAR-G protein coupling in the pathogenesis of the failing heart and point the way toward development of agents to inhibit βARK1 as a novel mode of therapy.
Adjustment of conformational flexibility is a key event in the thermal adaptation of proteins
Authors: Závodszky Péter , Kardos József , Svingor Ádám , Petsko Gregory A.
Published: July 2002

3-Isopropylmalate dehydrogenase (IPMDH, E.C. 1.1.1.85) from the thermophilic bacterium
Thermus thermophilus
HB8 is homologous to IPMDH from the mesophilic
Escherichia coli
, but has an approximately 17°C higher melting temperature. Its temperature optimum is 22–25°C higher than that of the
E. coli
enzyme; however, it is hardly active at room temperature. The increased conformational rigidity required to stabilize the thermophilic enzyme against heat denaturation might explain its different temperature-activity profile. Hydrogen/deuterium exchange studies were performed on this thermophilic-mesophilic enzyme pair to compare their conformational flexibilities. It was found that
Th. thermophilus
IPMDH is significantly more rigid at room temperature than
E. coli
IPMDH, whereas the enzymes have nearly identical flexibilities under their respective optimal working conditions, suggesting that evolutionary adaptation tends to maintain a “corresponding state” regarding conformational flexibility. These observations confirm that conformational fluctuations necessary for catalytic function are restricted at room temperature in the thermophilic enzyme, suggesting a close relationship between conformational flexibility and enzyme function.
Incomplete embryonic lethality and fatal neonatal hemorrhage caused by prothrombin deficiency in mice
Authors: Xue Jiachun , Wu Qingyu , Westfield Lisa A. , Tuley Elodee A. , Lu Deshun , Zhang Qing , Shim Kyuhwan , Zheng Xinglong , Sadler J. Evan
Published: July 2002

Deficiency of blood coagulation factor V or tissue factor causes the death of mouse embryos by 10.5 days of gestation, suggesting that part of the blood coagulation system is necessary for development. This function is proposed to require either generation of the serine protease thrombin and cell signaling through protease-activated receptors or an activity of tissue factor that is distinct from blood clotting. We find that murine deficiency of prothrombin clotting factor 2 (Cf2) was associated with the death of approximately 50% of Cf2
−/−
embryos by embryonic day 10.5 (E10.5), and surviving embryos had characteristic defects in yolk sac vasculature. Most of the remaining Cf2
−/−
embryos died by E15.5, but those surviving to E18.5 appeared normal. The rare Cf2
−/−
neonates died of hemorrhage on the first postnatal day. These studies suggest that a part of the blood coagulation system is adapted to perform a developmental function. Other mouse models show that the absence of platelets or of fibrinogen does not cause fetal wastage. Therefore, the role of thrombin in development may be independent of its effects on blood coagulation and instead may involve signal transduction on cells other than platelets.
Mitochondrial localization of a NADR-dependent isocitrate dehydrogenase isoenzyme by using the green fluorescent protein as a marker
Authors: Gálvez Susana , Roche Odile , Bismuth Evelyne , Brown Spencer , Gadal Pierre , Hodges Michael
Published: July 2002

In this work, we describe the isolation of a new cDNA encoding an NADP-dependent isocitrate dehydrogenase (ICDH). The nucleotide sequence in its 5′ region gives a deduced amino acid sequence indicative of a targeting peptide. However, even if this cDNA clearly encodes a noncytosolic ICDH, it is not possible to say from the targeting peptide sequence to which subcellular compartment the protein is addressed. To respond to this question, we have transformed tobacco plants with a construct containing the entire targeting signal-encoding sequence in front of a modified green fluorescent protein (GFP) gene. This construct was placed under the control of the cauliflower mosaic virus 35S promoter, and transgenic tobacco plants were regenerated. At the same time, and as a control, we also have transformed tobacco plants with the same construct but lacking the nucleotide sequence corresponding to the ICDH-targeting peptide, in which the GFP is retained in the cytoplasm. By optical and confocal microscopy of leaf epiderm and Western blot analyses, we show that the putative-targeting sequence encoded by the cDNA addresses the GFP exclusively into the mitochondria of plant cells. Therefore, we conclude that this cDNA encodes a mitochondrial ICDH.
Efficient adenoviral infection with IκBα reveals that macrophage tumor necrosis factor α production in rheumatoid arthritis is NF-κB dependent
Authors: Foxwell Brian , Browne Kylie , Bondeson Jan , Clarke Chris , de Martin Rainer , Brennan Fionula , Feldmann Marc
Published: July 2002

Tumor necrosis factor (TNF) α has been shown to be a major therapeutic target in rheumatoid arthritis with the success of anti-TNFα antibody clinical trials. Although signaling pathways leading to TNFα expression have been studied in some detail, there is evidence for considerable differences between individual cell types. This prompted us to investigate the intracellular signaling pathways that result in increased TNFα synthesis from macrophages in the diseased synovial joint tissue. Using an adenoviral system
in vitro
we report the successful delivery of genes to more than 95% of normal human macrophages. This permitted us to show, by using adenoviral transfer of IκBα, the natural inhibitor of NF-κB, that induction of TNFα in normal human macrophages by lipopolysaccharide, but not by some other stimuli, was inhibited by 80%. Furthermore the spontaneous production of TNFα from human rheumatoid joint cell cultures was inhibited by 75%, indicating that the NF-κB pathway is an essential step for TNFα synthesis in synovial macrophages and demonstrating that NF-κB should be an effective therapeutic target in this disease.
A phage display approach for rapid antibody humanization: Designed combinatorial V gene libraries
Authors: Rader Christoph , Cheresh David A. , Barbas Carlos F.
Published: July 2002

The development of a new strategy for antibody humanization is described. This strategy incorporates key recognition sequences from the parental rodent antibody into a phage display-based selection strategy. The original sequences of the third complementarity-determining regions (CDRs) of heavy and light chains, HCDR3 and LCDR3, were maintained and all other sequences were replaced by human sequences selected from phage-displayed antibody libraries. This approach was applied to the humanization of mouse mAb LM609 that is directed to human integrin αvβ3and has potential applicability in cancer therapy as an antiangiogenic agent. We demonstrate this approach (i) provides a rapid route for antibody humanization constraining the content of original mouse sequences in the final antibodies to the most hypervariable of the CDRs; (ii) generates several humanized versions with different sequences at the same time; (iii) results in affinities as high as or higher than the affinity of the original antibody; and (iv) retains the antigen and epitope specificity of the original antibody. The production of multiple humanized variants may present advantages in the selection of antibodies that are more readily expressed on a large scale and could be important in therapeutic regimens that call for long-term treatment with antibodies in which antiidiotypic responses might be avoided by administration of alternative antibodies.
The gene responsible for pseudohypoparathyroidism type Ib is paternally imprinted and maps in four unrelated kindreds to chromosome 20q13.3
Authors: Jüppner H. , Schipani E. , Bastepe M. , Cole D. E. C. , Lawson M. L. , Mannstadt M. , Hendy G. N. , Plotkin H. , Koshiyama H. , Koh T. , Crawford J. D. , Olsen B. R. , Vikkula M.
Published: July 2002

Hypocalcemia and hyperphosphatemia caused by parathyroid hormone (PTH)-resistance are the only discernible abnormalities in pseudohypoparathyroidism type Ib (PHP-Ib). Because mutations in the PTH/PTH-related peptide receptor, a plausible candidate gene, had been excluded previously, we conducted a genome-wide search with four PHP-Ib kindreds and established linkage to a small telomeric region on chromosome 20q, which contains the stimulatory G protein gene. We, furthermore, showed that the genetic defect is imprinted paternally and thus is inherited in the same mode as the PTH-resistant hypocalcemia in kindreds with PHP-Ia and/or pseudo-pseudohypoparathyroidism, two related disorders caused by different stimulatory G protein mutations.
20-Hydroxyeicosatetraenoic acid mediates calcium/calmodulin-dependent protein kinase II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells
Authors: Muthalif M. M. , Benter I. F. , Karzoun N. , Fatima S. , Harper J. , Uddin M. R. , Malik K. U.
Published: July 2002

Norepinephrine (NE) and angiotensin II (Ang II), by promoting extracellular Ca
2+
influx, increase Ca
2+
/calmodulin-dependent kinase II (CaMKII) activity, leading to activation of mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A
2
(cPLA
2
), resulting in release of arachidonic acid (AA) for prostacyclin synthesis in rabbit vascular smooth muscle cells. However, the mechanism by which CaMKII activates MAPK is unclear. The present study was conducted to determine the contribution of AA and its metabolites as possible mediators of CaMKII-induced MAPK activation by NE, Ang II, and epidermal growth factor (EGF) in vascular smooth muscle cells. NE-, Ang II-, and EGF-stimulated MAPK and cPLA
2
were reduced by inhibitors of cytochrome P450 (CYP450) and lipoxygenase but not by cyclooxygenase. NE-, Ang II-, and EGF-induced increases in Ras activity, measured by its translocation to plasma membrane, were abolished by CYP450, lipoxygenase, and farnesyltransferase inhibitors. An AA metabolite of CYP450, 20-hydroxyeicosatetraenoic acid (20-HETE), increased the activities of MAPK and cPLA
2
and caused translocation of Ras. These data suggest that activation of MAPK by NE, Ang II, and EGF is mediated by a signaling mechanism involving 20-HETE, which is generated by stimulation of cPLA
2
by CaMKII. Activation of Ras/MAPK by 20-HETE amplifies cPLA
2
activity and releases additional AA by a positive feedback mechanism. This mechanism of Ras/MAPK activation by 20-HETE may play a central role in the regulation of other cellular signaling molecules involved in cell proliferation and growth.
Working memory constrains human cooperation in the Prisoner’s Dilemma
Authors: Milinski Manfred , Wedekind Claus
Published: July 2002

Many problems in human society reflect the inability of selfish parties to cooperate. The “Iterated Prisoner’s Dilemma” has been used widely as a model for the evolution of cooperation in societies. Axelrod’s computer tournaments and the extensive simulations of evolution by Nowak and Sigmund and others have shown that natural selection can favor cooperative strategies in the Prisoner’s Dilemma. Rigorous empirical tests, however, lag behind the progress made by theorists. Clear predictions differ depending on the players’ capacity to remember previous rounds of the game. To test whether humans use the kind of cooperative strategies predicted, we asked students to play the iterated Prisoner’s Dilemma game either continuously or interrupted after each round by a secondary memory task (i.e., playing the game “Memory”) that constrained the students’ working-memory capacity. When playing without interruption, most students used “Pavlovian” strategies, as predicted, for greater memory capacity, and the rest used “generous tit-for-tat” strategies. The proportion of generous tit-for-tat strategies increased when games of Memory interfered with the subjects’ working memory, as predicted. Students who continued to use complex Pavlovian strategies were less successful in the Memory game, but more successful in the Prisoner’s Dilemma, which indicates a trade-off in memory capacity for the two tasks. Our results suggest that the set of strategies predicted by game theorists approximates human reality.
Inhibition of
HPV-16 E6/E7
immortalization of normal keratinocytes by hairpin ribozymes
Authors: Alvarez-Salas Luis M. , Cullinan Amy E. , Siwkowski Andrew , Hampel Arnold , DiPaolo Joseph A.
Published: July 2002

HPV-16 E6
and
E7
genes are required to efficiently immortalize a broad spectrum of cell types including cervical keratinocytes. Therefore, the
E6/E7
genes can be considered relevant targets for anti-cancer therapy. We produced several engineered hairpin (HP) ribozymes to specifically disrupt HPV-16 E6/E7 mRNA. After extensive biochemical characterization, one anti-E6 HP ribozyme (R434) was selected for
in vivo
testing because of its superior catalytic capabilities. When expressed in cis, R434 efficiently inhibited E6
in vitro
translation. Cis
-
expression of the HP ribozyme with
HPV-16 E6/E7
genes in normal human keratinocytes reduced the growth rate and prevented immortalization. RNA analysis by reverse transcription-PCR showed that E6/E7 transcripts were cleaved in post-transfected cells and virtually were eliminated after long term expression. Of interest, an inactive version of the HP also was able to significantly affect the immortalizing ability of E6/E7, probably through passive hybridization. The combination of passive and cleaving antisense RNA therefore is established as an effective inhibitor of HPV-16 E6/E7 immortalization.
Effects of feline immunodeficiency virus on astrocyte glutamate uptake: Implications for lentivirus-induced central nervous system diseases
Authors: Yu N. , Billaud J. N. , Phillips T. R.
Published: July 2002

Feline immunodeficiency virus (FIV) is a lentivirus of domestic cats that causes a spectrum of diseases remarkably similar to AIDS in HIV-infected humans. As part of this spectrum, both HIV-1 and FIV induce neurologic disorders. Because astrocytes are essential in maintaining the homeostasis of the central nervous system, we analyzed FIV for the ability to infect feline astrocytes. Through immunocytochemistry and reverse transcriptase activity, it was demonstrated that two molecular clones of FIV (FIV-34TF10 and FIV-PPR) produce a chronic low level productive infection of feline astrocyte cultures. To investigate the consequences of this infection, selected astrocyte functions were examined. Infection with FIV-34TF10 significantly decreased the ability of astrocytes to scavenge extracellular glutamate (with a peak inhibition of 74%). The effects of the infection did not appear to be a result of toxicity but rather were more selective in nature because the glucose uptake function of the infected astrocyte cultures was not altered. Our data demonstrate that FIV productively infected, at a low level, feline astrocyte cultures, and as a consequence of this infection, an important astroglial function was altered. These findings suggest that a chronic low grade infection of astrocytes may impair the ability of these cells to maintain homeostasis of the central nervous system that, in turn, may contribute to a neurodegenerative disease process that is often associated with lentivirus infections.
Solution structure of SpoIIAA, a phosphorylatable component of the system that regulates transcription factor σ
F
of
Bacillus subtilis
Authors: Kovacs Helena , Comfort David , Lord Matthew , Campbell Iain D. , Yudkin Michael D.
Published: July 2002

The establishment of differential gene expression in sporulating
Bacillus subtilis
involves four protein components, one of which, SpoIIAA, undergoes phosphorylation and dephosphorylation. We have used NMR spectroscopy to determine the solution structure of the nonphosphorylated form of SpoIIAA. The structure shows a fold consisting of a four-stranded β-sheet and four α-helices. Knowledge of the structure helps to account for the phenotype of several strains of
B. subtilis
that carry known
spoIIAA
mutations and should facilitate investigations of the conformational consequences of phosphorylation.
The oncogenic transforming potential of the passage of single α particles through mammalian cell nuclei
Authors: Miller Richard C. , Randers-Pehrson Gerhard , Geard Charles R. , Hall Eric J. , Brenner David J.
Published: July 2002

Domestic, low-level exposure to radon gas is considered a major environmental lung-cancer hazard involving DNA damage to bronchial cells by α particles from radon progeny. At domestic exposure levels, the relevant bronchial cells are very rarely traversed by more than one α particle, whereas at higher radon levels—at which epidemiological studies in uranium miners allow lung-cancer risks to be quantified with reasonable precision—these bronchial cells are frequently exposed to multiple α-particle traversals. Measuring the oncogenic transforming effects of exactly one α particle without the confounding effects of multiple traversals has hitherto been unfeasible, resulting in uncertainty in extrapolations of risk from high to domestic radon levels. A technique to assess the effects of single α particles uses a charged-particle microbeam, which irradiates individual cells or cell nuclei with predefined exact numbers of particles. Although previously too slow to assess the relevant small oncogenic risks, recent improvements in throughput now permit microbeam irradiation of large cell numbers, allowing the first oncogenic risk measurements for the traversal of exactly one α particle through a cell nucleus. Given positive controls to ensure that the dosimetry and biological controls were comparable, the measured oncogenicity from exactly one α particle was significantly lower than for a Poisson-distributed mean of one α particle, implying that cells traversed by multiple α particles contribute most of the risk. If this result applies generally, extrapolation from high-level radon risks (involving cellular traversal by multiple α particles) may overestimate low-level (involving only single α particles) radon risks.
Altered gene expression in striatal projection neurons in CB1 cannabinoid receptor knockout mice
Authors: Steiner Heinz , Bonner Tom I. , Zimmer Anne M. , Kitai Stephen T. , Zimmer Andreas
Published: July 2002

The basal ganglia, a brain structure critical for sensorimotor and motivational aspects of behavior, contain very high levels of CB1 cannabinoid receptors. These receptors are activated by endogenous lipophilic ligands, and they are thought to mediate behavioral effects of cannabinoid drugs. To evaluate the role of the endogenous cannabinoid system in the regulation of basal ganglia pathways, we have investigated the effects of targeted deletion of CB1 receptors on gene expression of various neuropeptides and transmitter-related enzymes in basal ganglia neurons. Mice without CB1 receptors are extremely hypoactive in a test for exploratory behavior (open-field test), showing markedly reduced locomotion and rearing. These CB1 mutants display significantly increased levels of substance P, dynorphin, enkephalin, and GAD 67 mRNAs in neurons of the two output pathways of the striatum that project to the substantia nigra and the globus pallidus. Our findings demonstrate that elimination of CB1 receptors results in behavioral abnormalities and functional reorganization of the basal ganglia.
Dual pathways for regulation of root branching by nitrate
Authors: Zhang Hanma , Jennings Andrea , Barlow Peter W. , Forde Brian G.
Published: July 2002

Root development is extremely sensitive to variations in nutrient supply, but the mechanisms are poorly understood. We have investigated the processes by which nitrate (NO
3
−
), depending on its availability and distribution, can have both positive and negative effects on the development and growth of lateral roots. When
Arabidopsis
roots were exposed to a locally concentrated supply of NO
3
−
there was no increase in lateral root numbers within the NO
3
−
-rich zone, but there was a localized 2-fold increase in the mean rate of lateral root elongation, which was attributable to a corresponding increase in the rate of cell production in the lateral root meristem. Localized applications of other N sources did not stimulate lateral root elongation, consistent with previous evidence that the NO
3
−
ion is acting as a signal rather than a nutrient. The
axr4
auxin-resistant mutant was insensitive to the stimulatory effect of NO
3
−
, suggesting an overlap between the NO
3
−
and auxin response pathways. High rates of NO
3
−
supply to the roots had a systemic inhibitory effect on lateral root development that acted specifically at the stage when the laterals had just emerged from the primary root, apparently delaying final activation of the lateral root meristem. A nitrate reductase-deficient mutant showed increased sensitivity to this systemic inhibitory effect, suggesting that tissue NO
3
−
levels may play a role in generating the inhibitory signal. We present a model in which root branching is modulated by opposing signals from the plant’s internal N status and the external supply of NO
3
−
.
Liver-derived insulin-like growth factor I (IGF-I) is the principal source of IGF-I in blood but is not required for postnatal body growth in mice
Authors: Sjögren Klara , Liu Jun-Li , Blad Kristina , Skrtic Stanko , Vidal Olle , Wallenius Ville , LeRoith Derek , Törnell Jan , Isaksson Olle G. P. , Jansson John-Olov , Ohlsson Claes
Published: July 2002

The body growth of animals is regulated by growth hormone and IGF-I. The classical theory of this regulation is that most IGF-I in the blood originates in the liver and that body growth is controlled by the concentration of IGF-I in the blood. We have abolished IGF-I production in the livers of mice by using the Cre/loxP recombination system. These mice demonstrated complete inactivation of the IGF-I gene in the hepatocytes. Although the liver accounts for less than 5% of body mass, the concentration of IGF-I in the serum was reduced by 75%. This finding confirms that the liver is the principal source of IGF-I in the blood. However, the reduction in serum IGF-I concentration had no discernible effect on postnatal body growth. We conclude that postnatal body growth is preserved despite complete absence of IGF-I production by the hepatocytes.
Natural
13
C abundance reveals trophic status of fungi and host-origin of carbon in mycorrhizal fungi in mixed forests
Authors: Högberg Peter , Plamboeck Agneta H. , Taylor Andrew F. S. , Fransson Petra M. A.
Published: July 2002

Fungi play crucial roles in the biogeochemistry of terrestrial ecosystems, most notably as saprophytes decomposing organic matter and as mycorrhizal fungi enhancing plant nutrient uptake. However, a recurrent problem in fungal ecology is to establish the trophic status of species in the field. Our interpretations and conclusions are too often based on extrapolations from laboratory microcosm experiments or on anecdotal field evidence. Here, we used natural variations in stable carbon isotope ratios (δ
13
C) as an approach to distinguish between fungal decomposers and symbiotic mycorrhizal fungal species in the rich sporocarp flora (our sample contains 135 species) of temperate forests. We also demonstrated that host-specific mycorrhizal fungi that receive C from overstorey or understorey tree species differ in their δ
13
C. The many promiscuous mycorrhizal fungi, associated with and connecting several tree hosts, were calculated to receive 57–100% of their C from overstorey trees. Thus, overstorey trees also support, partly or wholly, the nutrient-absorbing mycelia of their alleged competitors, the understorey trees.
Vasopressin contributes to hyperfiltration, albuminuria, and renal hypertrophy in diabetes mellitus: Study in vasopressin-deficient Brattleboro rats
Authors: Bardoux Pascale , Martin Hélène , Ahloulay Mina , Schmitt François , Bouby Nadine , Trinh-Trang-Tan Marie-Marcelle , Bankir Lise
Published: July 2002

Diabetic nephropathy represents a major complication of diabetes mellitus (DM), and the origin of this complication is poorly understood. Vasopressin (VP), which is elevated in type I and type II DM, has been shown to increase glomerular filtration rate in normal rats and to contribute to progression of chronic renal failure in 5/6 nephrectomized rats. The present study was thus designed to evaluate whether VP contributes to the renal disorders of DM. Renal function was compared in Brattleboro rats with diabetes insipidus (DI) lacking VP and in normal Long-Evans (LE) rats, with or without streptozotocin-induced DM. Blood and urine were collected after 2 and 4 weeks of DM, and creatinine clearance, urinary glucose and albumin excretion, and kidney weight were measured. Plasma glucose increased 3-fold in DM rats of both strains, but glucose excretion was ≈40% lower in DI-DM than in LE-DM, suggesting less intense metabolic disorders. Creatinine clearance increased significantly in LE-DM (
P
< 0.01) but failed to increase in DI-DM. Urinary albumin excretion more than doubled in LE-DM but rose by only 34% in DI-DM rats (
P
< 0.05). Kidney hypertrophy was also less intense in DI-DM than in LE-DM (
P
< 0.001). These results suggest that VP plays a critical role in diabetic hyperfiltration and albuminuria induced by DM. This hormone thus seems to be an additional risk factor for diabetic nephropathy and, thus, a potential target for prevention and/or therapeutic intervention.
Silicatein filaments and subunits from a marine sponge direct the polymerization of silica and silicones
in vitro
Authors: Cha Jennifer N. , Shimizu Katsuhiko , Zhou Yan , Christiansen Sean C. , Chmelka Bradley F. , Stucky Galen D. , Morse Daniel E.
Published: July 2002

Nanoscale control of the polymerization of silicon and oxygen determines the structures and properties of a wide range of siloxane-based materials, including glasses, ceramics, mesoporous molecular sieves and catalysts, elastomers, resins, insulators, optical coatings, and photoluminescent polymers. In contrast to anthropogenic and geological syntheses of these materials that require extremes of temperature, pressure, or pH, living systems produce a remarkable diversity of nanostructured silicates at ambient temperatures and pressures and at near-neutral pH. We show here that the protein filaments and their constituent subunits comprising the axial cores of silica spicules in a marine sponge chemically and spatially direct the polymerization of silica and silicone polymer networks from the corresponding alkoxide substrates
in vitro
, under conditions in which such syntheses otherwise require either an acid or base catalyst. Homology of the principal protein to the well known enzyme cathepsin L points to a possible reaction mechanism that is supported by recent site-directed mutagenesis experiments. The catalytic activity of the “silicatein” (
silica
pro
tein
) molecule suggests new routes to the synthesis of silicon-based materials.
An infection-based model of neurodevelopmental damage
Authors: Hornig Mady , Weissenböck Herbert , Horscroft Nigel , Lipkin W. Ian
Published: July 2002

Perinatal exposure to infectious agents and toxins is linked to the pathogenesis of neuropsychiatric disorders, but the mechanisms by which environmental triggers interact with developing immune and neural elements to create neurodevelopmental disturbances are poorly understood. We describe a model for investigating disorders of central nervous system development based on neonatal rat infection with Borna disease virus, a neurotropic noncytolytic RNA virus. Infection results in abnormal righting reflexes, hyperactivity, inhibition of open-field exploration, and stereotypic behaviors. Architecture is markedly disrupted in hippocampus and cerebellum, with reduction in granule and Purkinje cell numbers. Neurons are lost predominantly by apoptosis, as supported by increased mRNA levels for pro-apoptotic products (Fas, caspase-1), decreased mRNA levels for the anti-apoptotic bcl-x, andin situlabeling of fragmented DNA. Although inflammatory infiltrates are observed transiently in frontal cortex, glial activation (microgliosis > astrocytosis) is prominent throughout the brain and persists for several weeks in concert with increased levels of proinflammatory cytokine mRNAs (interleukins 1α, 1β, and 6 and tumor necrosis factor α) and progressive hippocampal and cerebellar damage. The resemblance of these functional and neuropathologic abnormalities to human neurodevelopmental disorders suggests the utility of this model for defining cellular, biochemical, histologic, and functional outcomes of interactions of environmental influences with the developing central nervous system.
A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window
Authors: Yrjänheikki Juha , Tikka Tiina , Keinänen Riitta , Goldsteins Gundars , Chan Pak H. , Koistinaho Jari
Published: July 2002

The only treatment of patients with acute ischemic stroke is thrombolytic therapy, which benefits only a fraction of stroke patients. Both human and experimental studies indicate that ischemic stroke involves secondary inflammation that significantly contributes to the outcome after ischemic insult. Minocycline is a semisynthetic second-generation tetracycline that exerts antiinflammatory effects that are completely separate from its antimicrobial action. Because tetracycline treatment is clinically well tolerated, we investigated whether minocycline protects against focal brain ischemia with a wide therapeutic window. Using a rat model of transient middle cerebral artery occlusion, we show that daily treatment with minocycline reduces cortical infarction volume by 76 ± 22% when the treatment is started 12 h before ischemia and by 63 ± 35% when started even 4 h after the onset of ischemia. The treatment inhibits morphological activation of microglia in the area adjacent to the infarction, inhibits induction of IL-1β-converting enzyme, and reduces cyclooxygenase-2 expression and prostaglandin E2production. Minocycline had no effect on astrogliosis or spreading depression, a wave of ionic transients thought to contribute to enlargement of cortical infarction. Treatment with minocycline may act directly on brain cells, because cultured primary neurons were also salvaged from glutamate toxicity. Minocycline may represent a prototype of an antiinflammatory compound that provides protection against ischemic stroke and has a clinically relevant therapeutic window.
Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins
Authors: Hardwick James S. , Kuruvilla Finny G. , Tong Jeffrey K. , Shamji Alykhan F. , Schreiber Stuart L.
Published: July 2002

The immunosuppressant rapamycin inhibits Tor1p and Tor2p (target of
rapamycin proteins), ultimately resulting in cellular responses
characteristic of nutrient deprivation through a mechanism involving
translational arrest. We measured the immediate transcriptional
response of yeast grown in rich media and treated with rapamycin to
investigate the direct effects of Tor proteins on nutrient-sensitive
signaling pathways. The results suggest that Tor proteins directly
modulate the glucose activation and nitrogen discrimination pathways
and the pathways that respond to the diauxic shift (including
glycolysis and the citric acid cycle). Tor proteins do not directly
modulate the general amino acid control, nitrogen starvation, or
sporulation (in diploid cells) pathways. Poor nitrogen quality
activates the nitrogen discrimination pathway, which is controlled by
the complex of the transcriptional repressor Ure2p and activator Gln3p.
Inhibiting Tor proteins with rapamycin increases the electrophoretic
mobility of Ure2p. The work presented here illustrates the coordinated
use of genome-based and biochemical approaches to delineate a cellular
pathway modulated by the protein target of a small molecule.
A serotonin transporter gene intron 2 polymorphic region, correlated with affective disorders, has allele-dependent differential enhancer-like properties in the mouse embryo
Authors: MacKenzie Alasdair , Quinn John
Published: July 2002

Polymorphic regions consisting of a variable number of tandem repeats within intron 2 of the gene coding for the serotonin transporter protein 5-HTT have been associated with susceptibility to affective disorders. We have cloned two of these intronic polymorphisms, Stin2.10 and Stin2.12, into an expression vector containing a heterologous minimal promoter and the bacterial
LacZ
reporter gene. These constructs were then used to produce transgenic mice. In embryonic day 10.5 embryos, both Stin2.10 and Stin2.12 produced consistent β-galactosidase expression in the embryonic midbrain, hindbrain, and spinal cord floor plate. However, we observed that the levels of β-galactosidase expression produced by both the Stin2.10 and Stin2.12 within the rostral hindbrain differed significantly at embryonic day 10.5. Our data suggest that these polymorphic variable number of tandem repeats regions act as transcriptional regulators and have allele-dependent differential enhancer-like properties within an area of the hindbrain where the 5-HTT gene is known to be transcribed at this stage of development.
Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes
Authors: Baron Udo , Schnappinger Dirk , Helbl Vera , Gossen Manfred , Hillen Wolfgang , Bujard Hermann
Published: July 2002

A regulatory system for the in-depth study of gene functions in higher eukaryotic cells has been developed. It is based on the tetracycline-controlled transactivators and reverse tTA, which were remodeled to discriminate efficiently between two different promoters. The system permits one to control reversibly the activity of two genes, or two alleles of a gene, in a mutually exclusive way, and also allows one to abrogate the activities of both. This dual regulatory circuit, which can be operated by a single effector substance such as doxycycline, overcomes limitations of conventional genetic approaches. The conditional mutants that can now be generated will be useful for the study of gene function
in vitro
and
in vivo
. In addition, the system may be of value for a variety of practical applications, including gene therapy.
Similarity in the catalysis of DNA breakage and rejoining by type IA and IIA DNA topoisomerases
Authors: Liu Qiyong , Wang James C.
Published: July 2002

Studies of yeast DNA topoisomerase II with various alanine-substitution mutations provide strong biochemical support of a recent hypothesis that the type IA and IIA DNA topoisomerases act similarly in their cleavage and rejoining of DNA. DNA breakage and rejoining by either a type IA or a type IIA enzyme are shown to involve cooperation between a DNA-binding domain containing the active-site tyrosine and a Rossmann fold containing several highly conserved acidic residues. For a homodimeric type IIA enzyme, cooperation occurs in trans: the active-site tyrosine in the DNA-binding domain of one protomer cooperates with several residues in the Rossmann fold as well as other regions of the other protomer.
Perception of solar UVB radiation by phytophagous insects: Behavioral responses and ecosystem implications
Authors: Mazza Carlos A. , Zavala Jorge , Scopel Ana L. , Ballaré Carlos L.
Published: July 2002

Most of our present knowledge about the impacts of solar UVB radiation on terrestrial ecosystems comes from studies with plants. Recently, the effects of UVB on the growth and survival of consumer species have begun to receive attention, but very little is known about UVB impacts on animal behavior. Here we report that manipulations of the flux of solar UVB received by field-grown soybean crops had large and consistent effects on the density of the thrips (
Caliothrips phaseoli
, Thysanoptera: Thripidae) populations that invaded the canopies, as well as on the amount of leaf damage caused by the insects. Solar UVB strongly reduced thrips herbivory. Thrips not only preferred leaves from plants that were not exposed to solar UVB over leaves from UVB-exposed plants in laboratory and field choice experiments, but they also appeared to directly sense and avoid exposure to solar UVB. Additional choice experiments showed that soybean leaf consumption by the late-season soybean worm
Anticarsia gemmatalis
(Lepidoptera: Noctuidae) was much less intense in leaves with even slight symptoms of an early thrips attack than in undamaged leaves. These experiments suggest that phytophagous insects can present direct and indirect behavioral responses to solar UVB. The indirect responses are mediated by changes in the plant host that are induced by UVB and, possibly, by other insects whose behavior is affected by UVB.
The crayfish plasma clotting protein: A vitellogenin-related protein responsible for clot formation in crustacean blood
Authors: Hall Martin , Wang Ruigong , van Antwerpen Rik , Sottrup-Jensen Lars , Söderhäll Kenneth
Published: July 2002

Coagulation in crayfish blood is based on the transglutaminase-mediated crosslinking of a specific plasma clotting protein. Here we report the cloning of the subunit of this clotting protein from a crayfish hepatopancreas cDNA library. The ORF encodes a protein of 1,721 amino acids, including a signal peptide of 15 amino acids. Sequence analysis reveals that the clotting protein is homologous to vitellogenins, which are proteins found in vitellogenic females of egg-laying animals. The clotting protein and vitellogenins are all lipoproteins and share a limited sequence similarity to certain other lipoproteins (e.g., mammalian apolipoprotein B and microsomal triglyceride transfer protein) and contain a stretch with similarity to the D domain of mammalian von Willebrand factor. The crayfish clotting protein is present in both sexes, unlike the female-specific vitellogenins. Electron microscopy was used to visualize individual clotting protein molecules and to study the transglutaminase-mediated clotting reaction. In the presence of an endogenous transglutaminase, the purified clotting protein molecules rapidly assemble into long, flexible chains that occasionally branch.
Angiostatin binds ATP synthase on the surface of human endothelial cells
Authors: Moser Tammy L. , Stack M. Sharon , Asplin Iain , Enghild Jan J. , Højrup Peter , Everitt Lorraine , Hubchak Susan , Schnaper H. William , Pizzo Salvatore V.
Published: July 2002

Angiostatin, a proteolytic fragment of plasminogen, is a potent antagonist of angiogenesis and an inhibitor of endothelial cell migration and proliferation. To determine whether the mechanism by which angiostatin inhibits endothelial cell migration and/or proliferation involves binding to cell surface plasminogen receptors, we isolated the binding proteins for plasminogen and angiostatin from human umbilical vein endothelial cells. Binding studies demonstrated that plasminogen and angiostatin bound in a concentration-dependent, saturable manner. Plasminogen binding was unaffected by a 100-fold molar excess of angiostatin, indicating the presence of a distinct angiostatin binding site. This finding was confirmed by ligand blot analysis of isolated human umbilical vein endothelial cell plasma membrane fractions, which demonstrated that plasminogen bound to a 44-kDa protein, whereas angiostatin bound to a 55-kDa species. Amino-terminal sequencing coupled with peptide mass fingerprinting and immunologic analyses identified the plasminogen binding protein as annexin II and the angiostatin binding protein as the α/β-subunits of ATP synthase. The presence of this protein on the cell surface was confirmed by flow cytometry and immunofluorescence analysis. Angiostatin also bound to the recombinant α-subunit of human ATP synthase, and this binding was not inhibited by a 2,500-fold molar excess of plasminogen. Angiostatin’s antiproliferative effect on endothelial cells was inhibited by as much as 90% in the presence of anti-α-subunit ATP synthase antibody. Binding of angiostatin to the α/β-subunits of ATP synthase on the cell surface may mediate its antiangiogenic effects and the down-regulation of endothelial cell proliferation and migration.
Clonal populations of hematopoietic cells with paroxysmal nocturnal hemoglobinuria genotype and phenotype are present in normal individuals
Authors: Araten David J. , Nafa Khedoudja , Pakdeesuwan Kriangsak , Luzzatto Lucio
Published: July 2002

In paroxysmal nocturnal hemoglobinuria (PNH), acquired somatic mutations in the
PIG-A
gene give rise to clonal populations of red blood cells unable to express proteins linked to the membrane by a glycosylphosphatidylinositol anchor. These proteins include the complement inhibitors CD55 and CD59, and this explains the hypersensitivity to complement of red cells in PNH patients, manifested by intravascular hemolysis. The factors that determine to what extent mutant clones expand have not yet been pinpointed; it has been suggested that existing PNH clones may have a conditional growth advantage depending on some factor (e.g., autoimmune) present in the marrow environment of PNH patients. Using flow cytometric analysis of granulocytes, we now have identified cells that have the PNH phenotype, at an average frequency of 22 per million (range 10–51 per million) in nine normal individuals. These rare cells were collected by flow sorting, and exons 2 and 6 of the
PIG-A
gene were amplified by nested PCR. We found
PIG-A
mutations in six cases: four missense, one frameshift, and one nonsense mutation. PNH red blood cells also were identified at a frequency of eight per million. Thus, small clones with
PIG-A
mutations exist commonly in normal individuals, showing clearly that
PIG-A
gene mutations are not sufficient for the development of PNH. Because
PIG-A
encodes an enzyme essential for the expression of a host of surface proteins, the
PIG-A
gene provides a highly sensitive system for the study of somatic mutations in hematopoietic cells.