bluuebunny/crossref_metadata_2025 huggingface dataset:
Elimination of ergovaline from a grass–
Neotyphodium
endophyte symbiosis by genetic modification of the endophyte
Authors: Panaccione Daniel G. , Johnson Richard D. , Wang Jinghong , Young Carolyn A. , Damrongkool Prapassorn , Scott Barry , Schardl Christopher L.
Published: July 2002

The fungal endophytes
Neotyphodium lolii
and
Neotyphodium
sp. Lp1 from perennial ryegrass (
Lolium perenne
), and related endophytes in other grasses, produce the ergopeptine toxin ergovaline, among other alkaloids, while also increasing plant fitness and resistance to biotic and abiotic stress. In the related fungus,
Claviceps purpurea
, the biosynthesis of ergopeptines requires the activities of two peptide synthetases, LPS1 and LPS2. A peptide synthetase gene hypothesized to be important for ergopeptine biosynthesis was identified in
C. purpurea
by its clustering with another ergot alkaloid biosynthetic gene,
dmaW
. Sequence analysis conducted independently of the research presented here indicates that this gene encodes LPS1 [Tudzynski, P., Holter, K., Correia, T., Arntz, C., Grammel, N. & Keller, U. (1999)
Mol. Gen. Genet.
261, 133–141]. We have cloned a similar peptide synthetase gene from
Neotyphodium lolii
and inactivated it by gene knockout in
Neotyphodium
sp. Lp1. The resulting strain retained full compatibility with its perennial ryegrass host plant as assessed by immunoblotting of tillers and quantitative PCR. However, grass–endophyte associations containing the knockout strain did not produce detectable quantities of ergovaline as analyzed by HPLC with fluorescence detection. Disruption of this gene provides a means to manipulate the accumulation of ergovaline in endophyte-infected grasses for the purpose of determining the roles of ergovaline in endophyte-associated traits and, potentially, for ameliorating toxicoses in livestock.
Molecular signals required for type III secretion and translocation of the
Xanthomonas campestris
AvrBs2 protein to pepper plants
Authors: Mudgett Mary Beth , Chesnokova Olga , Dahlbeck Doug , Clark Eszter T. , Rossier Ombeline , Bonas Ulla , Staskawicz Brian J.
Published: July 2002

Strains of
Xanthomonas campestris
pv.
vesicatoria
(
Xcv)
carrying
avrBs2
are specifically recognized by
Bs2
pepper plants, resulting in localized cell death and plant resistance.
Agrobacterium
-mediated transient expression of the
Xcv avrBs2
gene in plant cells results in
Bs2
-dependent cell death, indicating that the AvrBs2
protein alone is sufficient for the activation of disease
resistance-mediated cell death
in planta
. We now provide
evidence that AvrBs2 is secreted from
Xcv
and that
secretion is type III (
hrp
) dependent. N- and C-terminal
deletion analysis of AvrBs2 has identified the effector domain of
AvrBs2 recognized by
Bs2
pepper plants. By using a
truncated
Pseudomonas syringae
AvrRpt2 effector reporter
devoid of type III signal sequences, we have localized the minimal
region of AvrBs2 required for type III secretion in
Xcv.
Furthermore, we have identified the region of AvrBs2 required for both
type III secretion and translocation to host plants. The mapping of
AvrBs2 sequences sufficient for type III delivery also revealed the
presence of a potential mRNA secretion signal.
Personality traits and brain dopaminergic function in Parkinson's disease
Authors: Kaasinen Valtteri , Nurmi Elina , Bergman Jörgen , Eskola Olli , Solin Olof , Sonninen Pirkko , Rinne Juha O.
Published: July 2002

A distinctive personality type, characterized by introversion, inflexibility, and low novelty seeking, has been suggested to be associated with Parkinson's disease. To test the hypothesis that Parkinson's disease is associated with a specific dopamine-related personality type, the personality structures of 61 unmedicated Parkinson's disease patients and 45 healthy controls were examined. Additionally, in 47 Parkinson's disease patients, the dopaminergic function in the brain was directly measured with 6-[18F]fluoro-l-dopa (18F-dopa) positron emission tomography (PET) with MRI coregistration. The novelty-seeking personality score, supposedly associated with the parkinsonian personality, was slightly lower in the Parkinson's disease group compared with controls, but it did not have a significant relationship with18F-dopa uptake in any of the brain regions studied (r= −0.12 to 0.11,P> 0.15). The harm-avoidance personality score, associated with anxiety and depression, was clearly increased in patients with Parkinson's disease and it had a paradoxical, highly significant positive correlation with the18F-dopa uptake in the right caudate nucleus (r= 0.53,P= 0.04, Bonferroni corrected for 220 comparisons). Although the results of this study are not in disagreement with the concept of low-novelty-seeking personality type in Parkinson's disease, the personality type does not seem to be dopamine dependent. The correlation between the personality trait of harm avoidance and18F-dopa may reflect a specific feedback circuitry of neurotransmitters that is associated with negative emotionality in Parkinson's disease.
Recombinant bacillus Calmette–Guérin (BCG) vaccines expressing theMycobacterium tuberculosis30-kDa major secretory protein induce greater protective immunity against tuberculosis than conventional BCG vaccines in a highly susceptible animal model
Authors: Horwitz Marcus A. , Harth Günter , Dillon Barbara Jane , Masleša-Galić Saša
Published: July 2002

Tuberculosis (TB) continues to ravage humanity, causing 2 million deaths per year. A vaccine against TB more potent than the current live vaccine, bacillus Calmette–Guérin (BCG), is desperately needed. Using two commercially available strains of BCG as host strains, BCG Connaught and Tice, we have constructed two recombinant BCG vaccines stably expressing and secreting the 30-kDa major secretory protein ofMycobacterium tuberculosis(M. tb.), the primary causative agent of TB. We have tested the efficacy of the two strains in the highly susceptible guinea pig model of pulmonary TB, a model noteworthy for its close resemblance to human TB. Animals immunized with the recombinant BCG vaccines and challenged by aerosol with a highly virulent strain ofM. tb.had 0.5 logs fewerM. tb.bacilli in their lungs and 1 log fewer bacilli in their spleens on average than animals immunized with their parental conventional BCG vaccine counterparts. Statistically, these differences were highly significant. Paralleling these results, at necropsy, animals immunized with the recombinant BCG vaccines had fewer and smaller lesions in the lung, spleen, and liver and significantly less lung pathology than animals immunized with the parental BCG vaccines. The recombinant vaccines are the first vaccines against TB more potent than the current commercially available BCG vaccines, which were developed nearly a century ago.
Minocycline prevents nigrostriatal dopaminergic neurodegeneration in the MPTP model of Parkinson's disease
Authors: Du Yansheng , Ma Zhizhong , Lin Suizhen , Dodel Richard C. , Gao Feng , Bales Kelly R. , Triarhou Lazarose C. , Chernet Eyassu , Perry Ken W. , Nelson David L. G. , Luecke Susan , Phebus Lee A. , Bymaster Frank P. , Paul Steven M.
Published: July 2002

Parkinson's disease is a chronic neurodegenerative disorder
characterized by the loss of dopamine neurons in the substantia nigra,
decreased striatal dopamine levels, and consequent extrapyramidal motor
dysfunction. We now report that minocycline, a semisynthetic
tetracycline, recently shown to have neuroprotective effects in animal
models of stroke/ischemic injury and Huntington's disease, prevents
nigrostriatal dopaminergic neurodegeneration in the
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of
Parkinson's disease. Minocycline treatment also blocked dopamine
depletion in the striatum as well as in the nucleus accumbens after
MPTP administration. The neuroprotective effect of minocycline is
associated with marked reductions in inducible NO synthase (iNOS) and
caspase 1 expression.
In vitro
studies using primary
cultures of mesencephalic and cerebellar granule neurons (CGN) and/or
glia demonstrate that minocycline inhibits both
1-methyl-4-phenylpyridinium (MPP
+
)-mediated iNOS expression
and NO-induced neurotoxicity, but MPP
+
-induced
neurotoxicity is inhibited only in the presence of glia. Further,
minocycline also inhibits NO-induced phosphorylation of p38
mitogen-activated protein kinase (MAPK) in CGN and the p38 MAPK
inhibitor, SB203580, blocks NO toxicity of CGN. Our results suggest
that minocycline blocks MPTP neurotoxicity
in vivo
by
indirectly inhibiting MPTP/MPP
+
-induced glial iNOS
expression and/or directly inhibiting NO-induced neurotoxicity, most
likely by inhibiting the phosphorylation of p38 MAPK. Thus, NO appears
to play an important role in MPTP neurotoxicity. Neuroprotective
tetracyclines may be effective in preventing or slowing the progression
of Parkinson's and other neurodegenerative diseases.
Iron deficiency and iron excess damage mitochondria and mitochondrial DNA in rats
Authors: Walter Patrick B. , Knutson Mitchell D. , Paler-Martinez Andres , Lee Sonia , Xu Yu , Viteri Fernando E. , Ames Bruce N.
Published: July 2002

Approximately two billion people, mainly women and children, are iron deficient. Two studies examined the effects of iron deficiency and supplementation on rats. In study 1, mitochondrial functional parameters and mitochondrial DNA (mtDNA) damage were assayed in iron-deficient (≤5 μg/day) and iron-normal (800 μg/day) rats and in both groups after daily high-iron supplementation (8,000 μg/day) for 34 days. This dose is equivalent to the daily dose commonly given to iron-deficient humans. Iron-deficient rats had lower liver mitochondrial respiratory control ratios and increased levels of oxidants in polymorphonuclear-leukocytes, as assayed by dichlorofluorescein (
P
< 0.05). Rhodamine 123 fluorescence of polymorphonuclear-leukocytes also increased (
P
< 0.05). Lowered respiratory control ratios were found in daily high-iron-supplemented rats regardless of the previous iron status (
P
< 0.05). mtDNA damage was observed in both iron-deficient rats and rats receiving daily high-iron supplementation, compared with iron-normal rats (
P
< 0.05). Study 2 compared iron-deficient rats given high doses of iron (8,000 μg) either daily or every third day and found that rats given iron supplements every third day had less mtDNA damage on the second and third day after the last dose compared to daily high iron doses. Both inadequate and excessive iron (10 × nutritional need) cause significant mitochondrial malfunction. Although excess iron has been known to cause oxidative damage, the observation of oxidant-induced damage to mitochondria from iron deficiency has been unrecognized previously. Untreated iron deficiency, as well as excessive-iron supplementation, are deleterious and emphasize the importance of maintaining optimal iron intake.
Influence of anthropogenic aerosol on cloud optical depth and albedo shown by satellite measurements and chemical transport modeling
Authors: Schwartz Stephen E. , Harshvardhan  , Benkovitz Carmen M.
Published: July 2002

The Twomey effect of enhanced cloud droplet concentration, optical depth, and albedo caused by anthropogenic aerosols is thought to contribute substantially to radiative forcing of climate change over the industrial period. However, present model-based estimates of this indirect forcing are highly uncertain. Satellite-based measurements would provide global or near-global coverage of this effect, but previous efforts to identify and quantify enhancement of cloud albedo caused by anthropogenic aerosols in satellite observations have been limited, largely because of strong dependence of albedo on cloud liquid water path (LWP), which is inherently highly variable. Here we examine satellite-derived cloud radiative properties over two 1-week episodes for which a chemical transport and transformation model indicates substantial influx of sulfate aerosol from industrial regions of Europe or North America to remote areas of the North Atlantic. Despite absence of discernible dependence of optical depth or albedo on modeled sulfate loading, examination of the dependence of these quantities on LWP readily permits detection and quantification of increases correlated with sulfate loading, which are otherwise masked by variability of LWP, demonstrating brightening of clouds because of the Twomey effect on a synoptic scale. Median cloud-top spherical albedo was enhanced over these episodes, relative to the unperturbed base case for the same LWP distribution, by 0.02 to 0.15.
Principles of protein-protein interactions.
Authors: Jones S , Thornton J M
Published: July 2002

This review examines protein complexes in the Brookhaven Protein Databank to gain a better understanding of the principles governing the interactions involved in protein-protein recognition. The factors that influence the formation of protein-protein complexes are explored in four different types of protein-protein complexes--homodimeric proteins, heterodimeric proteins, enzyme-inhibitor complexes, and antibody-protein complexes. The comparison between the complexes highlights differences that reflect their biological roles.
Inhibition of immunoglobulin folding and secretion by dominant negative BiP ATPase mutants.
Authors: Hendershot L , Wei J , Gaut J , Melnick J , Aviel S , Argon Y
Published: July 2002

A group of resident ER proteins have been identified that are proposed to function as molecular chaperones. The best characterized of these is BiP/GRP78, an hsp70 homologue that binds peptides containing hydrophobic residues in vitro and unfolded or unassembled proteins in vivo. However, evidence that mammalian BiP plays a direct role in protein folding remains circumstantial. In this study, we examine how BiP interacts with a particular substrate, immunoglobulin light chain (lambda LC), during its folding. Wild-type hamster BiP and several well-characterized BiP ATPase mutants were used in transient expression experiments. We demonstrate that wild-type lambda LCs showed prolonged association with mutant BiP which inhibited their secretion. Both wild-type and mutant BiP bound only to unfolded and partially folded LCs. The wild-type BiP was released from the incompletely folded LCs, allowing them to fold and be secreted, whereas the mutant BiP was not released. As a result, the LCs that were bound to BiP mutants were unable to undergo complete disulfide bond formation and were retained in the ER. Our experiments suggest that LCs undergo both BiP-dependent and BiP-independent folding steps, demonstrating that both ATP binding and hydrolysis activities of BiP are essential for the completion of LC folding in vivo and reveal that BiP must release before disulfide bond formation can occur in that domain.
Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins.
Authors: Lima C D , Klein M G , Weinstein I B , Hendrickson W A
Published: July 2002

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.
Generation of mice with a 200-kb amyloid precursor protein gene deletion by Cre recombinase-mediated site-specific recombination in embryonic stem cells.
Authors: Li Z W , Stark G , Götz J , Rülicke T , Gschwind M , Huber G , Müller U , Weissmann C
Published: July 2002

Gene disruptions and deletions of up to 20kb have been generated by homologous recombination with appropriate targeting vectors in murine embryonic stem (ES) cells. Because we could not obtain a deletion of about 200 kb in the mouse amyloid precursor protein gene by the classical technique, we employed strategies involving the insertion of loxP sites upstream and downstream of the region to be deleted by homologous recombination and elicited excision of the loxP-flanked region by introduction of a Cre expression vector into the ES cells. In the first approach, the loxP sequences were inserted in two successive steps and after each step, ES cell clones were isolated and characterized. Deletion of the loxP-flanked sequence was accomplished by introducing the cre gene in a third step. In the second approach, ES cells containing the upstream loxP cassette were electroporated simultaneously with the downstream loxP targeting vector and the Cre expression plasmid. ES cells were obtained that gave rise to chimeric mice capable of germ-line transmission of the deleted amyloid precursor protein allele.
Pantropic retroviral vectors integrate and express in cells of the malaria mosquito, Anopheles gambiae.
Authors: Matsubara T , Beeman R W , Shike H , Besansky N J , Mukabayire O , Higgs S , James A A , Burns J C
Published: July 2002

The lack of efficient mechanisms for stable genetic transformation of medically important insects, such as anopheline mosquitoes, is the single most important impediment to progress in identifying novel control strategies. Currently available techniques for foreign gene expression in insect cells in culture lack the benefit of stable inheritance conferred by integration. To overcome this problem, a new class of pantropic retroviral vectors has been developed in which the amphotropic envelope is completely replaced by the G glycoprotein of vesicular stomatitis virus. The broadened host cell range of these particles allowed successful entry, integration, and expression of heterologous genes in cultured cells of Anopheles gambiae, the principle mosquito vector responsible for the transmission of over 100 million cases of malaria each year. Mosquito cells in culture infected with a pantropic vector expressing hygromycin phosphotransferase from the Drosophila hsp70 promoter were resistant to the antibiotic hygromycin B. Integrated provirus was detected in infected mosquito cell clones grown in selective media. Thus, pantropic retroviral vectors hold promise as a transformation system for mosquitoes in vivo.
Induction of long-term potentiation is associated with major ultrastructural changes of activated synapses.
Authors: Buchs P A , Muller D
Published: July 2002

Long-term potentiation (LTP), an increase in synaptic efficacy believed to underlie learning and memory mechanisms, has been proposed to involve structural modifications of synapses. Precise identification of the morphological changes associated with LTP has however been hindered by the difficulty in distinguishing potentiated or activated from nonstimulated synapses. Here we used a cytochemical method that allowed detection in CA1 hippocampus at the electron microscopy level of a stimulation-specific, D-AP5-sensitive accumulation of calcium in postsynaptic spines and presynaptic terminals following application of high-frequency trains. Morphometric analyses carried out 30-40 min after LTP induction revealed dramatic ultrastructural differences between labeled and nonlabeled synapses. The majority of labeled synapses (60%) exhibited perforated postsynaptic densities, whereas this proportion was only 20% in nonlabeled synaptic contacts. Labeled synaptic profiles were also characterized by a larger apposition zone between pre- and postsynaptic structures, longer postsynaptic densities, and enlarged spine profiles. These results add strong support to the idea that ultrastructural modifications and specifically an increase in perforated synapses are associated with LTP induction in field CA1 of hippocampus and they suggest that a majority of activated contacts may exhibit such changes.
The temporal lobe is a target of output from the basal ganglia.
Authors: Middleton F A , Strick P L
Published: July 2002

The basal ganglia are known to receive inputs from widespread regions of the cerebral cortex, such as the frontal, parietal, and temporal lobes. Of these cortical areas, only the frontal lobe is thought to be the target of basal ganglia output. One of the cortical regions that is a source of input to the basal ganglia is area TE, in inferotemporal cortex. This cortical area is thought to be critically involved in the recognition and discrimination of visual objects. Using retrograde transneuronal transport of herpes simplex virus type 1, we have found that one of the output nuclei of the basal ganglia, the substantia nigra pars reticulata, projects via the thalamus to TE. Thus, TE is not only a source of input to the basal ganglia, but also is a target of basal ganglia output. This result implies that the output of the basal ganglia influences higher order aspects of visual processing. In addition, we propose that dysfunction of the basal ganglia loop with TE leads to alterations in visual perception, including visual hallucinations.
Propagation of an attenuated virus by design: engineering a novel receptor for a noninfectious foot-and-mouth disease virus.
Authors: Rieder E , Berinstein A , Baxt B , Kang A , Mason P W
Published: July 2002

To gain entry into cells, viruses utilize a variety of different cell-surface molecules. Foot-and-mouth disease virus (FMDV) binds to cell-surface integrin molecules via an arginine-glycine-aspartic acid (RGD) sequence in capsid protein VP1. Binding to this particular cell-surface molecule influences FMDV tropism, and virus/receptor interactions appear to be responsible, in part, for selection of antigenic variants. To study early events of virus-cell interaction, we engineered an alternative and novel receptor for FMDV. Specifically, we generated a new receptor by fusing a virus-binding, single-chain antibody (scAb) to intracellular adhesion molecule 1 (ICAM1). Cells that are normally not susceptible to FMDV infection became susceptible after being transfected with DNA encoding the scAb/ICAM1 protein. An escape mutant (B2PD.3), derived with the mAb used to generate the genetically engineered receptor, was restricted for growth on the scAb/ICAM1 cells, but a variant of B2PD.3 selected by propagation on scAb/ICAM1 cells grew well on these cells. This variant partially regained wild-type sequence in the epitope recognized by the mAb and also regained the ability to be neutralize by the mAb. Moreover, RGD-deleted virions that are noninfectious in animals and other cell types grew to high titers and were able to form plaques on scAb/ ICAM1 cells. These studies demonstrate the first production of a totally synthetic cell-surface receptor for a virus. This novel approach will be useful for studying virus reception and for the development of safer vaccines against viral pathogens of animals and humans.
Rapid activation of Na+/H+ exchange by aldosterone in renal epithelial cells requires Ca2+ and stimulation of a plasma membrane proton conductance.
Authors: Gekle M , Golenhofen N , Oberleithner H , Silbernagl S
Published: July 2002

There is increasing evidence for an additional acute, nongenomic action of the mineralocorticoid hormone aldosterone on renal epithelial cells, leading to a two-step model of mineralocorticoid action on electrolyte excretion. We investigated the acute effect of aldosterone on intracellular free Ca2+ and on intracellular pH in an aldosterone-sensitive Madin-Darby canine kidney cell clone. Within seconds of application of aldosterone, but not of the glucocorticoid hydrocortisone, there was a 3-fold sustained increase of intracellular Ca2+ at a half-maximal concentration of 10(-10) mol/liter. Omission of extracellular Ca2+ prevented this hormone response. In the presence of extracellular Ca2+ aldosterone led to intracellular alkalinization. The Na+/H+ exchange inhibitor ethyl-isopropanol-amiloride (EIPA) prevented the aldosterone-induced alkalinization but not the aldosterone-induced increase of intracellular Ca2+. Omission of extracellular Ca2+ also prevented aldosterone-induced alkalinization. Instead, aldosterone led to a Zn(2+)-dependent intracellular acidification in the presence of EIPA, indicative of an increase of plasma membrane proton conductance. Under control conditions, Zn2+ prevented the aldosterone-induced alkalinization completely. We conclude that aldosterone stimulated net-entry of Ca2+ from the extracellular compartment and a plasma membrane H+ conductance as prerequisites for the stimulation of plasma membrane Na+/H+ exchange which in turn modulates K+ channel acitivity. It is probable that the aldosterone-sensitive H+ conductance maintains Na+/H+ exchange activity by providing an acidic environment in the vicinity of the exchanger. Thus, genomic action of aldosterone determines cellular transport equipment, whereas the nongenomic action regulates transporter activity that requires responses within seconds or minutes, which explains the rapid effects on electrolyte excretion.
Purification of telomerase from Euplotes aediculatus: requirement of a primer 3' overhang.
Authors: Lingner J , Cech T R
Published: July 2002

Telomerase is a ribonucleoprotein enzyme that uses its internal RNA moiety as a template for synthesis of telomeric repeats at chromosome ends. Here we report the purification of telomerase from Euplotes aediculatus by affinity chromatography with antisense 2'-O-methyl oligonucleotides, a method that was developed for small nuclear ribonucleoprotein particles (snRNPs). Elution of bound ribonucleoprotein from the antisense oligonucleotide under nondenaturing conditions was achieved by a novel approach, using a displacement oligonucleotide. Polypeptides of 120 kDa and 43 kDa (a doublet) copurify with the active telomerase and appear stoichiometric with telomerase RNA. A simple model for DNA end replication predicts that after semiconservative DNA replication, telomerase will extend the newly synthesized, blunt-ended leading strand. We show that purified Euplotes telomerase has no activity with blunt-ended primers. Instead, efficient extension requires 4 to 6 single-stranded nucleotides at the 3' end. Therefore, this model predicts the existence of other activities such as helicases or nucleases that generate a single-stranded 3' end from a blunt end, thus activating the end for telomerase extension.
Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach.
Authors: Burns A J , Lomax A E , Torihashi S , Sanders K M , Ward S M
Published: July 2002

The structural relationships between interstitial cells of Cajal (ICC), varicose nerve fibers, and smooth muscle cells in the gastrointestinal tract have led to the suggestion that ICC may be involved in or mediate enteric neurotransmission. We characterized the distribution of ICC in the murine stomach and found two distinct classes on the basis of morphology and immunoreactivity to antibodies against c-Kit receptors. ICC with multiple processes formed a network in the myenteric plexus region from corpus to pylorus. Spindle-shaped ICC were found within the circular and longitudinal muscle layers (IC-IM) throughout the stomach. The density of these cells was greatest in the proximal stomach. IC-IM ran along nerve fibers and were closely associated with nerve terminals and adjacent smooth muscle cells. IC-IM failed to develop in mice with mutations in c-kit. Therefore, we used W/W(V) mutants to test whether IC-IM mediate neural inputs in muscles of the gastric fundus. The distribution of inhibitory nerves in the stomachs of c-kit mutants was normal, but NO-dependent inhibitory neuro-regulation was greatly reduced. Smooth muscle tissues of W/W(V) mutants relaxed in response to exogenous sodium nitroprusside, but the membrane potential effects of sodium nitroprusside were attenuated. These data suggest that IC-IM play a critical serial role in NO-dependent neurotransmission: the cellular mechanism(s) responsible for transducing NO into electrical responses may be expressed in IC-IM. Loss of these cells causes loss of electrical responsiveness and greatly reduces responses to nitrergic nerve stimulation.
A test of the "jigsaw puzzle" model for protein folding by multiple methionine substitutions within the core of T4 lysozyme.
Authors: Gassner N C , Baase W A , Matthews B W
Published: July 2002

To test whether the structure of a protein is determined in a manner akin to the assembly of a jigsaw puzzle, up to 10 adjacent residues within the core of T4 lysozyme were replaced by methionine. Such variants are active and fold cooperatively with progressively reduced stability. The structure of a seven-methionine variant has been shown, crystallographically, to be similar to wild type and to maintain a well ordered core. The interaction between the core residues is, therefore, not strictly comparable with the precise spatial complementarity of the pieces of a jigsaw puzzle. Rather, a certain amount of give and take in forming the core structure is permitted. A simplified hydrophobic core sequence, imposed without genetic selection or computer-based design, is sufficient to retain native properties in a globular protein.
Discovery of genes involved with learning and memory: An experimental synthesis of Hirschian and Benzerian perspectives
Authors: Tully Tim
Published: July 2002

The biological bases of learning and memory are being revealed
today with a wide array of molecular approaches, most of which entail
the analysis of dysfunction produced by gene disruptions. This
perspective derives both from early “genetic dissections” of
learning in mutant
Drosophila
by Seymour Benzer and
colleagues and from earlier behavior-genetic analyses of learning and
in Diptera by Jerry Hirsch and coworkers. Three quantitative-genetic
insights derived from these latter studies serve as guiding principles
for the former. First, interacting polygenes underlie complex traits.
Consequently, learning/memory defects associated with single-gene
mutants can be quantified accurately only in equilibrated,
heterogeneous genetic backgrounds. Second, complex behavioral responses
will be composed of genetically distinct functional components. Thus,
genetic dissection of complex traits into specific biobehavioral
properties is likely. Finally, disruptions of genes involved with
learning/memory are likely to have pleiotropic effects. As a result,
task-relevant sensorimotor responses required for normal learning must
be assessed carefully to interpret performance in learning/memory
experiments. In addition, more specific conclusions will be obtained
from reverse-genetic experiments, in which gene disruptions are
restricted in time and/or space.
Intron positions correlate with module boundaries in ancient proteins
Authors: de Souza Sandro Jose , Long Manyuan , Schoenbach Lloyd , Roy Scott William , Gilbert Walter
Published: July 2002

We analyze the three-dimensional structure of proteins by a
computer program that finds regions of sequence that contain module
boundaries, defining a module as a segment of polypeptide chain bounded
in space by a specific given distance. The program defines a set of
“linker regions” that have the property that if an intron were to
be placed into each linker region, the protein would be dissected into
a set of modules all less than the specified diameter. We test a set of
32 proteins, all of ancient origin, and a corresponding set of 570
intron positions, to ask if there is a statistically significant excess
of intron positions within the linker regions. For 28-Å modules, a
standard size used historically, we find such an excess, with
P
< 0.003. This correlation is neither due to a
compositional or sequence bias in the linker regions nor to a surface
bias in intron positions. Furthermore, a subset of 20 introns, which
can be putatively identified as old, lies even more explicitly within
the linker regions, with
P
< 0.0003. Thus, there
is a strong correlation between intron positions and three-dimensional
structural elements of ancient proteins as expected by the
introns-early approach. We then study a range of module diameters and
show that, as the diameter varies, significant peaks of correlation
appear for module diameters centered at 21.7, 27.6, and 32.9 Å. These
preferred module diameters roughly correspond to predicted exon sizes
of 15, 22, and 30 residues. Thus, there are significant correlations
between introns, modules, and a quantized pattern of the lengths of
polypeptide chains, which is the prediction of the “Exon Theory of
Genes.”
Twenty-five coregulated transcripts define a sterigmatocystin gene cluster in Aspergillus nidulans.
Authors: Brown D W , Yu J H , Kelkar H S , Fernandes M , Nesbitt T C , Keller N P , Adams T H , Leonard T J
Published: July 2002

Sterigmatocystin (ST) and the aflatoxins (AFs), related fungal secondary metabolites, are among the most toxic, mutagenic, and carcinogenic natural products known. The ST biosynthetic pathway in Aspergillus nidulans is estimated to involve at least 15 enzymatic activities, while certain Aspergillus parasiticus, Aspergillus flavus, and Aspergillus nomius strains contain additional activities that convert ST to AF. We have characterized a 60-kb region in the A. nidulans genome and find it contains many, if not all, of the genes needed for ST biosynthesis. This region includes verA, a structural gene previously shown to be required for ST biosynthesis, and 24 additional closely spaced transcripts ranging in size from 0.6 to 7.2 kb that are coordinately induced only under ST-producing conditions. Each end of this gene cluster is demarcated by transcripts that are expressed under both ST-inducing and non-ST-inducing conditions. Deduced polypeptide sequences of regions within this cluster had a high percentage of identity with enzymes that have activities predicted for ST/AF biosynthesis, including a polyketide synthase, a fatty acid synthase (alpha and beta subunits), five monooxygenases, four dehydrogenases, an esterase, an 0-methyltransferase, a reductase, an oxidase, and a zinc cluster DNA binding protein. A revised system for naming the genes of the ST pathway is presented.
Peroxynitrite-mediated nitration of tyrosine residues in Escherichia coli glutamine synthetase mimics adenylylation: relevance to signal transduction.
Authors: Berlett B S , Friguet B , Yim M B , Chock P B , Stadtman E R
Published: July 2002

Treatment of Escherichia coli glutamine synthetase (GS) with peroxynitrite leads to nitration of some tyrosine residues and conversion of some methionine residues to methionine sulfoxide (MSOX) residues. Nitration, but not MSOX formation, is stimulated by Fe-EDTA. In the absence of Fe-EDTA, nitration of only one tyrosine residue per subunit of unadenylylated GS leads to changes in divalent cation requirement, pH-activity profile, affinity for ADP, and susceptibility to feedback inhibition by end products (tryptophan, AMP, CTP), whereas nitration of one tyrosine residue per subunit in the adenylylated GS leads to complete loss of catalytic activity. In the presence of Fe-EDTA, nitration is a more random process: nitration of five to six tyrosine residues per subunit is needed to convert unadenylylated GS to the adenylylated configuration. These results and the fact that nitration of tyrosine residues is an irreversible process serve notice that the regulatory function of proteins that undergo phosphorylation or adenylylation in signal transduction cascades might be seriously compromised by peroxynitrite-promoted nitration.
Human stanniocalcin: a possible hormonal regulator of mineral metabolism.
Authors: Olsen H S , Cepeda M A , Zhang Q Q , Rosen C A , Vozzolo B L
Published: July 2002

We have isolated a human cDNA clone encoding the mammalian homolog of stanniocalcin (STC), a calcium- and phosphate-regulating hormone that was first described in fishes where it functions in preventing hypercalcemia. STC has a unique amino acid sequence and, until now, has remained one of the few polypeptide hormones never described in higher vertebrates. Human STC (hSTC) was found to be 247 amino acids long and to share 73% amino acid sequence similarity with fish STC. Polyclonal antibodies to recombinant hSTC localized to a distinct cell type in the nephron tubule, suggesting kidney as a possible site of synthesis. Recombinant hSTC inhibited the gill transport of calcium when administered to fish and stimulated renal phosphate reabsorption in the rat. The evidence suggests that mammalian STC, like its piscine counterpart, is a regulator of mineral homeostasis.
Formation of chimeric DNA primer extension products by template switching onto an annealed downstream oligonucleotide.
Authors: Patel R , Lin M , Laney M , Kurn N , Rose S , Ullman E F
Published: July 2002

Oligodeoxynucleotide sequences are described that anneal to a template downstream of a priming site. During polymerase-catalyzed extension of the primer, the extending primer shifts from the original template to a segment of the annealed oligonucleotide that acts as an alternative template. The resulting chimeric extended primer has one segment that is complementary to the template and a second segment that is complementary to the oligonucleotide. The influence of the sequence elements of the oligonucleotide and the reaction conditions on template switching have been explored. The sequence requirements for template switching are compared to those for transposon excision.
Identification and characterization of a RING zinc finger gene (C-RZF) expressed in chicken embryo cells.
Authors: Tranque P , Crossin K L , Cirelli C , Edelman G M , Mauro V P
Published: July 2002

To identify changes in gene expression that occur in chicken embryo brain (CEB) cells as a consequence of their binding to the extracellular matrix molecule cytotactin/tenascin (CT/TN), a subtractive hybridization cloning strategy was employed. One of the cDNA clones identified was predicted to encode 381 amino acids and although it did not resemble any known sequences in the nucleic acid or protein data bases, it did contain the sequence motif for the cysteine-rich C3HC4 type of zinc finger, also known as a RING-finger. This sequence was therefore designated the chicken-RING zinc finger (C-RZF). In addition to the RING-finger, the C-RZF sequence also contained motifs for a leucine zipper, a nuclear localization signal, and a stretch of acidic amino acids similar to the activation domains of some transcription factors. Southern analysis suggested that C-RZF is encoded by a single gene. Northern and in situ hybridization analyses of E8 chicken embryo tissues indicated that expression of the C-RZF gene was restricted primarily to brain and heart. Western analysis of the nuclear and cytoplasmic fractions of chicken embryo heart cells and immunofluorescent staining of chicken embryo cardiocytes with anti-C-RZF antibodies demonstrated that the C-RZF protein was present in the nucleus. The data suggest that we have identified another member of the RING-finger family of proteins whose expression in CEB cells may be affected by CT/TN and whose nuclear localization and sequence motifs predict a DNA-binding function in the nucleus.
Involvement of specific macrophage-lineage cells surrounding arterioles in barrier and scavenger function in brain cortex.
Authors: Mato M , Ookawara S , Sakamoto A , Aikawa E , Ogawa T , Mitsuhashi U , Masuzawa T , Suzuki H , Honda M , Yazaki Y , Watanabe E , Luoma J , Yla-Herttuala S , Fraser I , Gordon S , Kodama T
Published: July 2002

The transport of solutes between blood and brain is regulated by a specific barrier. Capillary endothelial cells of brain are known to mediate barrier function and facilitate transport. Here we report that specific cells surrounding arterioles, known as Mato's fluorescent granular perithelial (FGP) cells or perivascular microglial cells, contribute to the barrier function. Immunohistochemical and in situ hybridization studies indicate that, in normal brain cortex, type I and type II macrophage scavenger receptors are expressed only in FGP/perivascular microglial cells, and surface markers of macrophage lineage are also detected on them. These cells mediate the uptake of macromolecules, including modified low density lipoprotein, horseradish peroxidase, and ferritin injected either into the blood or into the cerebral ventricles. Accumulation of scavenged materials with aging or after the administration of a high-fat diet results in the formation of honeycomb-like foam cells and the narrowing of the lumen of arterioles in the brain cortex. These results indicate involvement of FGP/perivascular microglial cells in the barrier and scavenger functions in the central nervous system.
Self-organized phase transitions in neural networks as a neural mechanism of information processing.
Authors: Hoshino O , Kashimori Y , Kambara T
Published: July 2002

Transitions between dynamically stable activity patterns imposed on an associative neural network are shown to be induced by self-organized infinitesimal changes in synaptic connection strength and to be a kind of phase transition. A key event for the neural process of information processing in a population coding scheme is transition between the activity patterns encoding usual entities. We propose that the infinitesimal and short-term synaptic changes based on the Hebbian learning rule are the driving force for the transition. The phase transition between the following two dynamical stable states is studied in detail, the state where the firing pattern is changed temporally so as to itinerate among several patterns and the state where the firing pattern is fixed to one of several patterns. The phase transition from the pattern itinerant state to a pattern fixed state may be induced by the Hebbian learning process under a weak input relevant to the fixed pattern. The reverse transition may be induced by the Hebbian unlearning process without input. The former transition is considered as recognition of the input stimulus, while the latter is considered as clearing of the used input data to get ready for new input. To ensure that information processing based on the phase transition can be made by the infinitesimal and short-term synaptic changes, it is absolutely necessary that the network always stays near the critical state corresponding to the phase transition point.
Characterization of the gene cluster of high-molecular-mass nitrile hydratase (H-NHase) induced by its reaction product in Rhodococcus rhodochrous J1.
Authors: Komeda H , Kobayashi M , Shimizu S
Published: July 2002

The 4.6-kb region 5'-upstream from the gene encoding a cobalt-containing and amide-induced high molecular mass-nitrile hydratase (H-NHase) from Rhodococcus rhodochrous J1 was found to be required for the expression of the H-NHase gene with a host-vector system in a Rhodococcus strain. Sequence analysis has revealed that there are at least five open reading frames (H-ORF1 approximately 5) in addition to H-NHase alpha- and beta-subunit genes. Deletion of H-ORF1 and H-ORF2 resulted in decrease of NHase activity, suggesting a positive regulatory role of both ORFs in the expression of the H-NHase gene. H-ORF1 showed significant similarity to a regulatory protein, AmiC, which is involved in regulation of amidase expression by binding an inducer amide in Pseudomonas aeruginosa. H-ORF4, which has been found to be uninvolved in regulation of H-NHase expression by enzyme assay for its deletion transformant and Northern blot analysis for R. rhodochrous J1, showed high similarity to transposases from insertion sequences of several bacteria. Determination of H-NHase activity and H-NHase mRNA levels in R. rhodochrous J1 has indicated that the expression of the H-NHase gene is regulated by an amide at the transcriptional level. These findings suggest the participation of H-ORF4 (IS1164) in the organization of the H-NHase gene cluster and the involvement of H-ORF1 in unusual induction mechanism, in which H-NHase is formed by amides (the products in the NHase reaction), but not by nitriles (the substrates).
Folding of the N-terminal, ligand-binding region of integrin α-subunits into a β-propeller domain
Authors: Springer Timothy A.
Published: July 2002

The N-terminal ≈440 aa of integrin α subunits contain seven
sequence repeats. These are predicted here to fold into a β-propeller
domain. A homologous domain from the enzyme phosphatidylinositol
phospholipase D is predicted to have the same fold. The domains contain
seven four-stranded β-sheets arranged in a torus around a
pseudosymmetry axis. The trimeric G-protein β subunit (G beta)
appears to be the most closely related β-propeller. Integrin ligands
and a putative Mg
2+
ion are predicted to bind to the upper
face of the β-propeller. This face binds substrates in β-propeller
enzymes and is used by the G protein β subunit to bind the G protein
α subunit. The integrin α subunit I domain, which is structurally
homologous to the G protein α subunit, is tethered to the top of the
β-propeller domain by a hinge that may allow movement of the domains
relative to one another. The Ca
2+
-binding motifs in
integrin α subunits are on the lower face of the
β-propeller.
Messenger RNA deadenylylation precedes decapping in mammalian cells
Authors: Couttet P. , Fromont-Racine M. , Steel D. , Pictet R. , Grange T.
Published: July 2002

In yeast, the major mRNA degradation pathway is initiated by poly(A) tail shortening that triggers mRNA decapping. The mRNA is then degraded by 5′-to-3′ exonucleolysis. In mammalian cells, even though poly(A) tail shortening also precedes mRNA degradation, the degradation pathway has not been elucidated. We have used a reverse transcription–PCR approach that relies on mRNA circularization to measure the poly(A) tail length of four mammalian mRNAs. This approach allows for the simultaneous analysis of the 5′ and 3′ ends of the same mRNA molecule. For all four mRNAs analyzed, this strategy permitted us to demonstrate the existence of small amounts of decapped mRNA species which have a shorter poly(A) tail than their capped counterparts. Kinetic analysis of one of these mRNAs indicates that the decapped species with a short poly(A) tail are mRNA degradation products. Therefore, our results indicate that decapping is preceded by a shortening of the poly(A) tail in mammalian cells, as it is in yeast, suggesting that this mRNA degradation pathway is conserved throughout eukaryotic evolution.