bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning of human adenosine kinase cDNA: sequence similarity to microbial ribokinases and fructokinases.
Authors: Spychala J , Datta N S , Takabayashi K , Datta M , Fox I H , Gribbin T , Mitchell B S
Published: July 2002

Adenosine kinase catalyzes the phosphorylation of adenosine to AMP and hence is a potentially important regulator of extracellular adenosine concentrations. Despite extensive characterization of the kinetic properties of the enzyme, its primary structure has never been elucidated. Full-length cDNA clones encoding catalytically active adenosine kinase were obtained from lymphocyte, placental, and liver cDNA libraries. Corresponding mRNA species of 1.3 and 1.8 kb were noted on Northern blots of all tissues examined and were attributable to alternative polyadenylylation sites at the 3' end of the gene. The encoding protein consists of 345 amino acids with a calculated molecular size of 38.7 kDa and does not contain any sequence similarities to other well-characterized mammalian nucleoside kinases, setting it apart from this family of structurally and functionally related proteins. In contrast, two regions were identified with significant sequence identity to microbial ribokinase and fructokinases and a bacterial inosine/guanosine kinase. Thus, adenosine kinase is a structurally distinct mammalian nucleoside kinase that appears to be akin to sugar kinases of microbial origin.
Zn2+ interaction with Alzheimer amyloid beta protein calcium channels.
Authors: Arispe N , Pollard H B , Rojas E
Published: July 2002

The Alzheimer disease 40-residue amyloid beta protein (AbetaP[1-40]) forms cation-selective channels across acidic phospholipid bilayer membranes with spontaneous transitions over a wide range of conductances ranging from 40 to 4000 pS. Zn2+ has been reported to bind to AbetaP[1-40] with high affinity, and it has been implicated in the formation of amyloid plaques. We now report the functional consequences of such Zn2+ binding for the AbetaP[1-40] channel. Provided the AbetaP[1-40] channel is expressed in the low conductance (<400 pS) mode, Zn2+ blocks the open channel in a dose- dependent manner. For AbetaP[1-40] channels in the giant conductance mode (>400 pS), Zn2+ doses in the millimolar range were required to exert substantial blockade. The Zn2+ chelator o-phenanthroline reverses the blockade. We also found that Zn2+ modulates AbetaP[1-40] channel gating and conductance only from one side of the channel. These data are consistent with predictions of our recent molecular modeling studies on AbetaP[1-40] channels indicating asymmetric Zn(2+)-AbetaP[1-40] interactions at the entrance to the pore.
Transposon tools for recombinant DNA manipulation: characterization of transcriptional regulators from yeast, Xenopus, and mouse.
Authors: Morgan B A , Conlon F L , Manzanares M , Millar J B , Kanuga N , Sharpe J , Krumlauf R , Smith J C , Sedgwick S G
Published: July 2002

Transposon Tn1000 has been adapted to deliver novel DNA sequences for manipulating recombinant DNA. The transposition procedure for these "tagged" Tn1000s is simple and applicable to most plasmids in current use. For yeast molecular biology, tagged Tn1000s introduce a variety of yeast selective markers and replication origins into plasmids and cosmids. In addition, the beta-globin minimal promoter and lacZ gene of Tn(beta)lac serve as a mobile reporter of eukaryotic enhancer activity. In this paper, Tn(beta)lac was used to localize a mouse HoxB-complex enhancer in transgenic mice. Other tagged transposons create Gal4 DNA-binding-domain fusions, in either Escherichia coli or yeast plasmids, for use in one- and two-hybrid tests of transcriptional activation and protein-protein interaction, respectively. With such fusions, the Saccharomyces cerevisiae Swi6 G1/S-phase transcription factor and the Xenopus laevis Pintallavis developmental regulator are shown to activate transcription. Furthermore, the same transposon insertions also facilitated mapping of the Swi6 and Pintallavis domains responsible for transcriptional activation. Thus, as well as introducing novel sequences, tagged transposons share the numerous other applications of transposition such as producing insertional mutations, creating deletion series, or serving as mobile primer sites for DNA sequencing.
Mapping protein-protein interactions by affinity-directed mass spectrometry.
Authors: Zhao Y , Muir T W , Kent S B , Tischer E , Scardina J M , Chait B T
Published: July 2002

A precise and rapid method for identifying sites of interaction between proteins was demonstrated; the basis of the method is direct mass spectrometric readout from the complex to determine the specific components of the proteins that interact--a method termed affinity-directed mass spectrometry. The strategy was used to define the region of interaction of a protein growth factor with a monoclonal antibody. A combination of proteolytic digestion and affinity-directed mass spectrometry was used to rapidly determine the approximate location of a continuous binding epitope within the growth factor. The precise boundaries of the binding epitope were determined by affinity-directed mass spectrometric analysis of sets of synthetic peptide ladders that span the approximate binding region. In addition to the mapping of such linear epitopes, affinity-directed mass spectrometry can be applied to the mapping of other types of molecule-molecule contacts, including ligand-receptor and protein-oligonucleotide interactions.
Efficient photoreceptor-targeted gene expression
in vivo
by recombinant adeno-associated virus
Authors: Flannery John G. , Zolotukhin Sergei , Vaquero M. Isabel , LaVail Matthew M. , Muzyczka Nicholas , Hauswirth William W.
Published: July 2002

We describe a general approach for achieving efficient and cell type-specific expression of exogenous genes in photoreceptor cells of the mammalian retina. Recombinant adeno-associated virus (rAAV) vectors were used to transfer the bacterial
lacZ
gene or a synthetic green fluorescent protein gene (
gfp
) to mouse or rat retinas after injection into the subretinal space. Using a proximal murine rod opsin promoter (+86 to −385) to drive expression, reporter gene product was found exclusively in photoreceptors, not in any other retinal cell type or in the adjacent retinal pigment epithelium. GFP-expressing photoreceptors typically encompassed 10–20% of the total retinal area after a single 2-μl injection. Photoreceptors were transduced with nearly 100% efficiency in the region directly surrounding the injection site. We estimate approximately 2.5 million photoreceptors were transduced as a result of the single subretinal inoculation. This level of gene transfer and expression suggests the feasibility of genetic therapy for retinal disease. The
gfp
-containing rAAV stock was substantially free of both adenovirus and wild-type AAV, as judged by plaque assay and infectious center assay, respectively. Thus, highly purified, helper virus-free rAAV vectors can achieve high-frequency tissue-specific transduction of terminally differentiated, postmitotic photoreceptor cells.
Transcriptional abnormality in myotonic dystrophy affects
DMPK
but not neighboring genes
Authors: Hamshere Marion G. , Newman Emma E. , Alwazzan Madawi , Athwal Balwinder S. , Brook J. David
Published: July 2002

Myotonic dystrophy (DM) is caused by the expansion of a trinucleotide repeat, CTG, in the 3′ untranslated region of a protein kinase gene,
DMPK
. We set out to determine what effect this expanded repeat has on RNA processing. The subcellular fractionation of RNA and the separate analysis of
DMPK
transcripts from each allele reveals that transcripts from expanded
DMPK
alleles are retained within the nucleus and are absent from the cytoplasm of DM cell lines. The nuclear retention of
DMPK
transcripts occurs above a critical threshold between 80 and 400 CTGs. Further analysis of the nuclear RNA reveals an apparent reduction in the proportion of expansion-derived DMPK transcripts after poly(A)
+
selection. Quantitative analysis of RNA also indicates that although the level of cytoplasmic
DMPK
transcript is altered in DM patients, the levels of transcripts from
59
and
DMAHP
, two genes that immediately flank
DMPK
, are unaffected in DM cell lines.
Transcription and activity of antioxidant enzymes after ionizing irradiation in radiation-resistant and radiation-sensitive mice
Authors: Hardmeier Rosmarie , Hoeger Harald , Fang-Kircher Susanne , Khoschsorur Ali , Lubec Gert
Published: July 2002

The involvement of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase in radiobiological processes has been described at the enzyme activity level. We irradiated radiation-resistant (RR) and radiation-sensitive (RS) mice and studied antioxidant enzymes at the transcriptional and activity level. In addition, aromatic hydroxylation and lipid peroxidation parameters were determined to study radiation resistance at the oxidation level. RS BALB/c/J Him mice and RR C3H He/Him mice were whole-body-irradiated with x-rays at 2, 4, and 6 Gy and killed 5, 15, and 30 min after irradiation. mRNA was isolated from liver and hybridized with probes for antioxidant enzymes and β-actin as a housekeeping gene control. Antioxidant enzyme activities were determined by standard assays. Parameters for aromatic hydroxylation (
o
-tyrosine) and lipid peroxidation (
malondialdehyde
) were determined by HPLC methods. Antioxidant transcription was unchanged in contrast to antioxidant activities; SOD and CAT activities were elevated within 15 min in RR animals but not in RS mice, at all doses studied. Glutathione peroxidase activity was not different between RR and RS mice and was only moderately elevated after irradiation. No significant differences were found between RR and RS animals at the oxidation level, although a radiation dose-dependent increase of oxidation products was detected in both groups. We found that ionizing irradiation led to increased antioxidant activity only minutes after irradiation in the absence of increased transcription of these antioxidant enzymes. RR animals show higher antioxidant enzyme activities than do RS mice, but oxidation products are comparable in RS and RR mice. As unchanged transcription of antioxidant enzymes could not have been responsible for the increased antioxidant enzyme activities, preformed antioxidant enzymes should have been released by the irradiation process. This would be in agreement with previous studies of preformed, stored SOD. The finding of higher SOD and CAT activities in RR than in RS animals could point to a role for these antioxidant enzymes for the process of radiation sensitivity.
Modeling the control of DNA replication in fission yeast
Authors: Novak Bela , Tyson John J.
Published: July 2002

A central event in the eukaryotic cell cycle is the decision to commence DNA replication (S phase). Strict controls normally operate to prevent repeated rounds of DNA replication without intervening mitoses (“endoreplication”) or initiation of mitosis before DNA is fully replicated (“mitotic catastrophe”). Some of the genetic interactions involved in these controls have recently been identified in yeast. From this evidence we propose a molecular mechanism of “Start” control in
Schizosaccharomyces pombe
. Using established principles of biochemical kinetics, we compare the properties of this model in detail with the observed behavior of various mutant strains of fission yeast:
wee1
−
(size control at Start),
cdc13
Δ and
rum1

OP

(endoreplication), and
wee1
−
rum1
Δ (rapid division cycles of diminishing cell size). We discuss essential features of the mechanism that are responsible for characteristic properties of Start control in fission yeast, to expose our proposal to crucial experimental tests.
Neuronal death in the central nervous system demonstrates a non-fibrin substrate for plasmin
Authors: Tsirka Stella E. , Bugge Thomas H. , Degen Jay L. , Strickland Sidney
Published: July 2002

Mice deficient for plasminogen exhibit a variety of pathologies, all of which examined to date are reversed when the animals are also made fibrin(ogen) deficient. These results suggested that the predominant, and perhaps exclusive, physiological role of plasminogen is clearance of fibrin. Plasminogen-deficient mice also display resistance to excitotoxin-induced neurodegeneration, in contrast with wild-type mice, which are sensitive. Based on the genetic interaction between plasminogen and fibrinogen, we investigated whether resistance to neuronal cell death in the plasminogen-deficient mice is dependent on fibrin(ogen). Unexpectedly, mice lacking both plasminogen and fibrinogen are resistant to neurodegeneration to levels comparable to plasminogen-deficient mice. Therefore, plasmin acts on substrates other than fibrin during experimental neuronal degeneration, and may function similarly in other pathological settings in the central nervous system.
Targeted disruption of the mouse gene encoding steroidogenic acute regulatory protein provides insights into congenital lipoid adrenal hyperplasia
Authors: Caron Kathleen M. , Soo Shiu-Ching , Wetsel William C. , Stocco Douglas M. , Clark Barbara J. , Parker Keith L.
Published: July 2002

An essential component of regulated steroidogenesis is the translocation of cholesterol from the cytoplasm to the inner mitochondrial membrane where the cholesterol side-chain cleavage enzyme carries out the first committed step in steroidogenesis. Recent studies showed that a 30-kDa mitochondrial phosphoprotein, designated steroidogenic acute regulatory protein (StAR), is essential for this translocation. To allow us to explore the roles of StAR in a system amenable to experimental manipulation and to develop an animal model for the human disorder lipoid congenital adrenal hyperplasia (lipoid CAH), we used targeted gene disruption to produce StAR knockout mice. These StAR knockout mice were indistinguishable initially from wild-type littermates, except that males and females had female external genitalia. After birth, they failed to grow normally and died from adrenocortical insufficiency. Hormone assays confirmed severe defects in adrenal steroids—with loss of negative feedback regulation at hypothalamic–pituitary levels—whereas hormones constituting the gonadal axis did not differ significantly from levels in wild-type littermates. Histologically, the adrenal cortex of StAR knockout mice contained florid lipid deposits, with lesser deposits in the steroidogenic compartment of the testis and none in the ovary. The sex-specific differences in gonadal involvement support a two-stage model of the pathogenesis of StAR deficiency, with trophic hormone stimulation inducing progressive accumulation of lipids within the steroidogenic cells and ultimately causing their death. These StAR knockout mice provide a useful model system in which to determine the mechanisms of StAR’s essential roles in adrenocortical and gonadal steroidogenesis.
Mitochondrial mutational spectra in human cells and tissues
Authors: Khrapko Konstantin , Coller Hilary A. , André Paulo C. , Li Xiao-Cheng , Hanekamp John S. , Thilly William G.
Published: July 2002

We have found that human organs such as colon, lung, and muscle, as well as their derived tumors, share nearly all mitochondrial hotspot point mutations. Seventeen hotspots, primarily G → A and A → G transitions, have been identified in the mitochondrial sequence of base pairs 10,030–10,130. Mutant fractions increase with the number of cell generations in a human B cell line, TK6, indicating that they are heritable changes. The mitochondrial point mutation rate appears to be more than two orders of magnitude higher than the nuclear point mutation rate in TK6 cells and in human tissues. The similarity of the hotspot sets
in vivo
and
in vitro
leads us to conclude that human mitochondrial point mutations in the sequence studied are primarily spontaneous in origin and arise either from DNA replication error or reactions of DNA with endogenous metabolites. The predominance of transition mutations and the high number of hotspots in this short sequence resembles spectra produced by DNA polymerases
in vitro
.
Variable and tissue-specific hormone resistance in heterotrimeric G
s
protein α-subunit (G
s
α) knockout mice is due to tissue-specific imprinting of the G
s
α gene
Authors: Yu Shuhua , Yu Dawen , Lee Eric , Eckhaus Michael , Lee Randy , Corria Zakia , Accili Domenico , Westphal Heiner , Weinstein Lee S.
Published: July 2002

Albright hereditary osteodystrophy (AHO), an autosomal dominant disorder characterized by short stature, obesity, and skeletal defects, is associated with heterozygous inactivating mutations of
GNAS1
, the gene encoding the heterotrimeric G protein α-subunit (G
s
α) that couples multiple receptors to the stimulation of adenylyl cyclase. It has remained unclear why only some AHO patients present with multihormone resistance and why AHO patients demonstrate resistance to some hormones [e.g., parathyroid hormone (PTH)] but not to others (e.g., vasopressin), even though all activate adenylyl cyclase. We generated mice with a null allele of the mouse homolog
Gnas.
Homozygous G
s
deficiency is embryonically lethal. Heterozygotes with maternal (m−/+) and paternal (+/p−) inheritance of the
Gnas
null allele have distinct phenotypes, suggesting that
Gnas
is an imprinted gene. PTH resistance is present in m−/+, but not +/p−, mice. G
s
α expression in the renal cortex (the site of PTH action) is markedly reduced in m−/+ but not in +/p− mice, demonstrating that the
Gnas
paternal allele is imprinted in this tissue.
Gnas
is also imprinted in brown and white adipose tissue. The maximal physiological response to vasopressin (urinary concentrating ability) is normal in both m−/+ and +/p− mice and
Gnas
is not imprinted in the renal inner medulla (the site of vasopressin action). Tissue-specific imprinting of
Gnas
is likely the mechanism for variable and tissue-specific hormone resistance in these mice and a similar mechanism might explain the variable phenotype in AHO.
Visual representations during saccadic eye movements
Authors: Moore Tirin , Tolias Andreas S. , Schiller Peter H.
Published: July 2002

In normal vision, shifts of attention are usually followed by saccadic eye movements. Neurons in extrastriate area V4 are modulated by focal attention when eye movements are withheld, but they also respond in advance of visually guided saccadic eye movements. We have examined the visual selectivity of saccade-related responses of area V4 neurons in monkeys making delayed eye movements to receptive field stimuli of varying orientation. This task did not require the monkey to attend to orientation
per se
but merely to foveate the receptive field stimulus. We present evidence that the presaccadic enhancement exhibited by V4 neurons, quite separate from the response at stimulus onset, is a resurgent visual representation that seems as selective as the response is when the stimulus first appears. The presaccadic enhancement appears to provide a strengthening of a decaying featural representation immediately before an eye movement is directed to visual targets. We suggest that this reactivation provides a mechanism by which a clear perception of the saccade goal can be maintained during the execution of the saccade, perhaps for the purpose of establishing continuity across eye movements.
Activation of the genetically defined m1 muscarinic receptor potentiates
N
-methyl-
d
-aspartate (NMDA) receptor currents in hippocampal pyramidal cells
Authors: Marino Michael J. , Rouse Susan T. , Levey Allan I. , Potter Lincoln T. , Conn P. Jeffrey
Published: July 2002

Evidence suggests that cholinergic input to the hippocampus plays an important role in learning and memory and that degeneration of cholinergic terminals in the hippocampus may contribute to the memory loss associated with Alzheimer’s disease. One of the more prominent effects of cholinergic agonists on hippocampal physiology is the potentiation of
N
-methyl-
d
-aspartate (NMDA)-receptor currents by muscarinic agonists. Here, we employ traditional pharmacological reagents as well as m1-toxin, an m1 antagonist with unprecedented selectivity, to demonstrate that this potentiation of NMDA-receptor currents in hippocampal CA1 pyramidal cells is mediated by the genetically defined m1 muscarinic receptor. Furthermore, we demonstrate the colocalization of the m1 muscarinic receptor and the NR1a NMDA receptor subunit at the electron microscopic level, indicating a spatial relationship that would allow for physiological interactions between these two receptors. This work demonstrates that the m1-muscarinic receptor gene product modulates excitatory synaptic transmission, and it has important implications in the study of learning and memory as well as the design of drugs to treat neurodegenerative diseases such as Alzheimer’s.
Isolation and one-step preparation of A2E and
iso
-A2E, fluorophores from human retinal pigment epithelium
Authors: Parish Craig A. , Hashimoto Masaru , Nakanishi Koji , Dillon James , Sparrow Janet
Published: July 2002

Age-related macular degeneration, a major cause of blindness for which no satisfactory treatments exist, leads to a gradual decrease in central high acuity vision. The accumulation of fluorescent materials, called lipofuscin, in retinal pigment epithelial cells of the aging retina is most pronounced in the macula. One of the fluorophores of retinal pigment epithelial lipofuscin has been characterized as A2E, a pyridinium bis-retinoid, which is derived from two molecules of vitamin A aldehyde and one molecule of ethanolamine. An investigation aimed at optimizing the
in vitro
synthesis of A2E has resulted in the one-step biomimetic preparation of this pigment in 49% yield, readily producing more than 50 mg in one step. These results have allowed for the optimization of HPLC conditions so that nanogram quantities of A2E can be detected from extracts of tissue samples. By using 5% of the extract from individual aged human eyes, this protocol has led to the quantification of A2E and the characterization of
iso
-A2E, a new A2E double bond isomer; all-
trans
-retinol and 13-
cis-
retinol also have been identified in these HPLC chromatograms. Exposure of either A2E or
iso
-A2E to light gives rise to 4:1 A2E:
iso
-A2E equilibrium mixtures, similar to the composition of these two pigments in eye extracts. A2E and
iso
-A2E may exhibit surfactant properties arising from their unique wedge-shaped structures.
Targeted disruption of mouse long-chain acyl-CoA dehydrogenase gene reveals crucial roles for fatty acid oxidation
Authors: Kurtz David M. , Rinaldo Piero , Rhead William J. , Tian Liqun , Millington David S. , Vockley Jerry , Hamm Doug A. , Brix Amy E. , Lindsey J. Russell , Pinkert Carl A. , O’Brien William E. , Wood Philip A.
Published: July 2002

Abnormalities of fatty acid metabolism are recognized to play a significant role in human disease, but the mechanisms remain poorly understood. Long-chain acyl-CoA dehydrogenase (LCAD) catalyzes the initial step in mitochondrial fatty acid oxidation (FAO). We produced a mouse model of LCAD deficiency with severely impaired FAO. Matings between LCAD +/− mice yielded an abnormally low number of LCAD +/− and −/− offspring, indicating frequent gestational loss. LCAD −/− mice that reached birth appeared normal, but had severely reduced fasting tolerance with hepatic and cardiac lipidosis, hypoglycemia, elevated serum free fatty acids, and nonketotic dicarboxylic aciduria. Approximately 10% of adult LCAD −/− males developed cardiomyopathy, and sudden death was observed in 4 of 75 LCAD −/− mice. These results demonstrate the crucial roles of mitochondrial FAO and LCAD
in vivo
.
CREB-binding protein in androgen receptor-mediated signaling
Authors: Aarnisalo Piia , Palvimo Jorma J. , Jänne Olli A.
Published: July 2002

CREB-binding protein (CBP) is a transcriptional coregulator that interacts with different DNA binding proteins and components of the general transcription machinery. CBP enhanced androgen receptor (AR)-dependent transcription under transient transfection conditions in CV-1 cells. The ligand binding domain (LBD) and residues 38–296 of the N-terminal region of AR are not required because the activity of a receptor mutant devoid of these domains was augmented by coexpressed CBP. There is physical interaction between AR and CBP
in vivo
, as judged by coimmunoprecipitation experiments from cell extracts. Consistent with the role of CBP as a coactivator for AR, the 12S E1A adenoviral protein that inactivates CBP function strongly inhibited AR-dependent transactivation. Exogenous CBP was also capable of overcoming the inhibitory effect of AR on AP-1 activity and diminished the mutual transcriptional repression between AR and NF-κB (RelA). Collectively, these data imply that transcriptional interference between AR and AP-1 or NF-κB is mediated, at least in part, through competition for intracellular CBP and that this coactivator serves as an integrator between androgen-mediated and other signaling pathways.
A serine cluster prevents recycling of the V2 vasopressin receptor
Authors: Innamorati Giulio , Sadeghi Hamid M. , Tran Nathaniel T. , Birnbaumer Mariel
Published: July 2002

Receptor recycling plays a critical role in the regulation of cellular responsiveness to environmental stimuli. Agonist-promoted phosphorylation of G protein-coupled receptors has been related to their desensitization, internalization, and sequestration. Dephosphorylation of internalized G protein-coupled receptors by cytoplasmic phosphatases has been shown to be pH-dependent, and it has been postulated to be necessary for receptors to recycle to the cell surface. The internalized V2 vasopressin receptor (V2R) expressed in HEK 293 cells is an exception to this hypothesis because it does not recycle to the plasma membrane for hours after removal of the ligand. Because this receptor is phosphorylated only by G protein-coupled receptor kinases (GRKs), the relationship between recycling and GRK-mediated phosphorylation was examined. A nonphosphorylated V2R, truncated upstream of the GRK phosphorylation sites, rapidly returned to the cell surface after removal of vasopressin. Less-drastic truncations of V2R revealed the presence of multiple phosphorylation sites and suggested a key role for a serine cluster present at the C terminus. Replacement of any one of Ser-362, Ser-363, or Ser-364 with Ala allowed quantitative recycling of full-length V2R without affecting the extent of internalization. Examination of the stability of phosphate groups incorporated into the recycling S363A mutant V2Rs revealed that the recycling receptor was dephosphorylated after hormone withdrawal, whereas the wild-type V2R was not, providing molecular evidence for the hypothesis that GRK sites must be dephosphorylated prior to receptor recycling. These experiments uncovered a role for GRK phosphorylation in intracellular sorting and revealed a GRK-dependent anchoring domain that blocks V2R recycling.
Data transferability from model organisms to human beings: Insights from the functional genomics of the
flightless
region of
Drosophila
Authors: Maleszka R. , de Couet H. G. , Miklos George L. Gabor
Published: July 2002

At what biological levels are data from single-celled organisms akin to a Rosetta stone for multicellular ones? To examine this question, we characterized a saturation-mutagenized 67-kb region of the
Drosophila
genome by gene deletions, transgenic rescues, phenotypic dissections, genomic and cDNA sequencing, bio-informatic analysis, reverse transcription–PCR studies, and evolutionary comparisons. Data analysis using cDNA/genomic DNA alignments and bio-informatic algorithms revealed 12 different predicted proteins, most of which are absent from bacterial databases, half of which are absent from
Saccharomyces cerevisiae
, and nearly all of which have relatives in
Caenorhabditis elegans
and
Homo sapiens
. Gene order is not evolutionarily conserved; the closest relatives of these genes are scattered throughout the yeast, nematode, and human genomes. Most gene expression is pleiotropic, and deletion studies reveal that a morphological phenotype is seldom observed when these genes are removed from the genome. These data pinpoint some general bottlenecks in functional genomics, and they reveal the acute emerging difficulties with data transferability above the levels of genes and proteins, especially with complex human phenotypes. At these higher levels the Rosetta stone analogy has almost no applicability. However, newer transgenic technologies in
Drosophila
and
Mus
, combined with coherency pattern analyses of gene networks, and synthetic neural modeling, offer insights into organismal function. We conclude that industrially scaled robogenomics in model organisms will have great impact if it can be realistically linked to epigenetic analyses of human variation and to phenotypic analyses of human diseases in different genetic backgrounds.
Direct evidence for a predominantly exolytic processive mechanism for depolymerization of heparin-like glycosaminoglycans by heparinase I
Authors: Ernst Steffen , Rhomberg Andrew J. , Biemann Klaus , Sasisekharan Ram
Published: July 2002

Heparinase I from
Flavobacterium heparinum
has important uses for elucidating the complex sequence heterogeneity of heparin-like glycosaminoglycans (HLGAGs). Understanding the biological function of HLGAGs has been impaired by the limited methods for analysis of pure or mixed oligosaccharide fragments. Here, we use methodologies involving MS and capillary electrophoresis to investigate the sequence of events during heparinase I depolymerization of HLGAGs. In an initial step, heparinase I preferentially cleaves exolytically at the nonreducing terminal linkage of the HLGAG chain, although it also cleaves internal linkages at a detectable rate. In a second step, heparinase I has a strong preference for cleaving the same substrate molecule processively, i.e., to cleave the next site toward the reducing end of the HLGAG chain. Computer simulation showed that the experimental results presented here from analysis of oligosaccharide degradation were consistent with literature data for degradation of polymeric HLGAG by heparinase I. This study presents direct evidence for a predominantly exolytic and processive mechanism of depolymerization of HLGAG by heparinase I.
Mechanism of platelet inhibition by nitric oxide:
In vivo
phosphorylation of thromboxane receptor by cyclic GMP-dependent protein kinase
Authors: Wang Guang-Rong , Zhu Yan , Halushka Perry V. , Lincoln Thomas M. , Mendelsohn Michael E.
Published: July 2002

Nitric oxide (NO) is a potent vasodilator and inhibitor of platelet activation. NO stimulates production of cGMP and activates cGMP-dependent protein kinase (G kinase), which by an unknown mechanism leads to inhibition of Gα
q
-phospholipase C-inositol 1,4,5-triphosphate signaling and intracellular calcium mobilization for several important agonists, including thromboxane A
2
(TXA
2
). To explore the mechanism of platelet inhibition by NO, activation of platelet TXA
2
receptors in the presence of cGMP was studied. The nonhydrolyzable analog 8-bromo-cyclic GMP (8-Br-cGMP) potently inhibited activation of the TXA
2
-specific GTPase in platelet membranes in a concentration-dependent fashion, suggesting that G kinase catalyzes the phosphorylation of some proximal component of the receptor–G protein signaling pathway. Nanomolar concentrations of G kinase were found to catalyze the phosphorylation of platelet TXA
2
receptors
in vitro
, but not Gα
q
copurifying with the TXA
2
receptors in these experiments. Using immunoaffinity methods,
in vivo
phosphorylation of TXA
2
receptors by cyclic GMP was demonstrated from
32
P-labeled cells treated with 8-Br-cGMP. Peptide mapping studies of
in vivo
phosphorylated TXA
2
receptors demonstrated cGMP mediates phosphorylation of the carboxyl terminus of the TXA
2
receptor. G kinase also catalyzed the phosphorylation of peptides corresponding to the cytoplasmic tails of both α and β forms of the receptor but not control peptide or a peptide corresponding to the third intracytoplasmic loop of the TXA
2
receptor. These data identify TXA
2
receptors as cGMP-dependent protein kinase substrates and support a novel mechanism for the inhibition of cell function by NO in which activation of G kinase inhibits signaling by G protein-coupled receptors by catalyzing their phosphorylation.
Evidence of hominid-like precision grip capability in the hand of the Miocene ape
Oreopithecus
Authors: Moyà-Solà Salvador , Köhler Meike , Rook Lorenzo
Published: July 2002

Functional and allometric analyses of the hand of the late Miocene ape
Oreopithecus bambolii
(Tuscany, Italy) reveal a series of features that reflect an improved grasping capability including firm pad-to-pad precision gripping that apes are unable to perform. Related features such as hand length, relative thumb length, a deep and large insertion area for the tendon of the long thumb flexor, and the form of the metacarpal 2/capitate articulation are not present in extant or fossil apes. In these features, the
Oreopithecus
hand closely matches the pattern of early hominids, presumably as a response to similar functional demands.
NS5A, a nonstructural protein of hepatitis C virus, binds growth factor receptor-bound protein 2 adaptor protein in a Src homology 3 domain/ligand-dependent manner and perturbs mitogenic signaling
Authors: Tan Seng-Lai , Nakao Haruhisa , He Yupeng , Vijaysri Sangeetha , Neddermann Petra , Jacobs Bertram L. , Mayer Bruce J. , Katze Michael G.
Published: July 2002

Although hepatitis C virus (HCV) infection is an emerging global epidemic causing severe liver disorders, the molecular mechanisms of HCV pathogenesis remain elusive. The NS5A nonstructural protein of HCV contains several proline-rich sequences consistent with Src homology (SH) 3-binding sites found in cellular signaling molecules. Here, we demonstrate that NS5A specifically bound to growth factor receptor-bound protein 2 (Grb2) adaptor protein. Immunoblot analysis of anti-Grb2 immune complexes derived from HeLa S3 cells infected with a recombinant vaccinia virus (VV) expressing NS5A revealed an interaction between NS5A and Grb2in vivo. An inactivating point mutation in the N-terminal SH3 domain, but not in the C-terminal SH3 domain, of Grb2 displayed significant diminished binding to NS5A. However, the same mutation in both SH3 regions completely abrogated Grb2 binding to NS5A, implying that the two SH3 domains bind in cooperative fashion to NS5A. Further, mutational analysis of NS5A assigned the SH3-binding region to a proline-rich motif that is highly conserved among HCV genotypes. Importantly, phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) was inhibited in HeLa S3 cells infected with NS5A-expressing recombinant VV but not recombinant VV control. Additionally, HeLa cells stably expressing NS5A were refractory to ERK1/2 phosphorylation induced by exogenous epidermal growth factor. Moreover, the coupling of NS5A to Grb2 in these cells was induced by epidermal growth factor stimulation. Therefore, NS5A may function to perturb Grb2-mediated signaling pathways by selectively targeting the adaptor. These findings highlight a viral interceptor of cellular signaling with potential implications for HCV pathogenesis.
A central role for thymic emigrants in peripheral T cell homeostasis
Authors: Berzins S. P. , Godfrey D. I. , Miller J. F. A. P. , Boyd R. L.
Published: July 2002

After initial seeding by thymic emigrants, homeostatic regulation of the T cell pool has been thought to occur entirely within the periphery. Here we report that the degree of thymic emigration directly affects the number and the CD4/CD8 ratio of peripheral T cells. We demonstrate that the increase in T cell pool size caused by the engraftment of 2, 6, or 9 thymic lobes correlates almost exactly with the number of emigrants exported from those grafts in the previous 3 weeks, regardless of how long the graft has been in place. The extent of the increase supports the concept of a 3-week period after thymic export in which emigrant T cells are exempt from peripheral T cell homeostasis. This apparent exclusion of recent thymic emigrants from the niche-based regulation of peripheral T cell numbers ensures repertoire turnover throughout adult life and provides the basis of a direct and previously unrecognized role for the thymus in the regulation of peripheral T cell homeostasis.
RNAs that bind and change the permeability of phospholipid membranes
Authors: Khvorova Anastasia , Kwak Yong-Geun , Tamkun Michael , Majerfeld Irene , Yarus Michael
Published: July 2002

The RNA world hypothesis presumes that RNA will be competent for varied essential cellular functions. One such indispensable cell function is regulation of membrane permeability. Though this was not a known RNA activity, selection–amplification yielded RNAs that bound phosphatidylcholine:cholesterol liposomes. At least eight distinct, ≈95-mer sequences bind well to the outside of the lipid bilayer, though randomized sequences had no such activity. No distinct sequence motif for lipid binding was found. However, truncation of one such RNA shows that a smaller, 44-nucleotide irregular RNA hairpin is an active membrane binding domain. Bound RNA increases the permeability of liposomes to
22
Na
+
. In addition, using voltage clamp technique, four individual RNAs increase the ion permeability of the plasma membrane of cultured human cells. The existence of multiple sequences that bind membranes and provoke permeability changes suggests that these may be elementary RNA functions that could be selected
in vivo
.
Functional analysis of an eye specific enhancer of the
eyeless
gene in
Drosophila
Authors: Hauck Bernd , Gehring Walter J. , Walldorf Uwe
Published: July 2002

The development of the
Drosophila
compound eye requires the function of a set of evolutionarily conserved genes. Among these, the
Drosophila Pax-6
gene
eyeless
(
ey
) plays a major role.
ey
has been considered a master control gene of eye development in the animal kingdom because targeted expression of
ey
and vertebrate as well as invertebrate homologs lead to the formation of ectopic eyes in
Drosophila
. We demonstrate that an intron of the
ey
gene contains an enhancer that regulates the eye specific expression of the gene in the eye disc primordia of embryos and in the eye imaginal discs of third instar larvae. Moreover, a 212-bp enhancer element is necessary and sufficient for the enhancer function. It is partially conserved in
Drosophila hydei
and contains putative Pax-6 Paired domain binding sites. We show that several binding sites are required for the eye specific expression, and, therefore, we propose a
Pax-6
-like molecule to be a positive transactivator for the eye specific
ey
expression. This transactivator recently has been identified as
twin of eyeless
, the second
Pax-6
gene in
Drosophila
.
The effect of antibody-dependent enhancement on the transmission dynamics and persistence of multiple-strain pathogens
Authors: Ferguson Neil , Anderson Roy , Gupta Sunetra
Published: July 2002

Cross-reactive antibodies produced by a mammalian host during infection by a particular microparasitic strain usually have the effect of reducing the probability of the host being infected by a different, but closely related, pathogen strain. Such cross-reactive immunological responses thereby induce between-strain competition within the pathogen population. However, in some cases such as dengue virus, evidence suggests that cross-reactive antibodies act to enhance rather than restrict the severity of a subsequent infection by another strain. This cooperative mechanism is thought to explain why pre-existing immunity to dengue virus is an important risk factor for the development of severe disease (i.e., dengue shock syndrome and dengue hemorrhagic fever). In this paper, we explore the effect of antibody-dependent enhancement on the transmission dynamics of multistrain pathogen populations. We show that enhancement frequently may generate complex and persistent cyclical or chaotic epidemic behavior. Furthermore, enhancement acts to permit the coexistence of all strains where in its absence only one or a subset would persist.
Hierarchies and logarithmic oscillations in the temporal relaxation patterns of proteins and other complex systems
Authors: Metzler Ralf , Klafter Joseph , Jortner Joshua
Published: July 2002

Logarithmic oscillations superimposed on the temporal relaxation patterns of complex systems are considered from the standpoint of their hierarchical origin. We propose that a closer examination of experimental data should reveal logarithmic oscillations in systems that are characterized by a hierarchical structure of their dynamical degrees of freedom. On that footing, a new methodology of data analysis is proposed that may prove important for the dynamics of protein folding and of conformational fluctuations in proteins in which the relevant time scales of the dynamical evolution underlying the relaxation kinetics can be deduced from these oscillations.
Humoral response to herpes simplex virus is complement-dependent
Authors: Da Costa Xavier J. , Brockman Mark A. , Alicot Elisabeth , Ma Minghe , Fischer Michael B. , Zhou Xioaning , Knipe David M. , Carroll Michael C.
Published: July 2002

The complement system represents a cascade of serum proteins, which provide a major effector function in innate immunity. Recent studies have revealed that complement links innate and adaptive immunity via complement receptors CD21/CD35 in that it enhances the B cell memory response to noninfectious protein antigens introduced i.v. To examine the importance of complement for immune responses to virus infection in a peripheral tissue, we compared the B cell memory response of mice deficient in complement C3, C4, or CD21/CD35 with wild-type controls. We found that the deficient mice failed to generate a normal memory response, which is characterized by a reduction in IgG antibody and germinal centers. Thus, complement is important not only in the effector function of innate immunity but also in the stimulation of memory B cell responses to viral-infected cell antigens in both blood and peripheral tissues.
The MinC component of the division site selection system in
Escherichia coli
interacts with FtsZ to prevent polymerization
Authors: Hu Zonglin , Mukherjee Amit , Pichoff Sebastien , Lutkenhaus Joe
Published: July 2002

Positioning of the Z ring at the midcell site in
Escherichia coli
is assured by the
min
system, which masks polar sites through topological regulation of MinC, an inhibitor of division. To study how MinC inhibits division, we have generated a MalE-MinC fusion that retains full biological activity. We find that MalE-MinC interacts with FtsZ and prevents polymerization without inhibiting FtsZ's GTPase activity. MalE-MinC19 has reduced ability to inhibit division, reduced affinity for FtsZ, and reduced ability to inhibit FtsZ polymerization. These results, along with MinC localization, suggest that MinC rapidly oscillates between the poles of the cell to destabilize FtsZ filaments that have formed before they mature into polar Z rings.
Type 2 diabetes: Evidence for linkage on chromosome 20 in 716 Finnish affected sib pairs
Authors: Ghosh Soumitra , Watanabe Richard M. , Hauser Elizabeth R. , Valle Timo , Magnuson Victoria L. , Erdos Michael R. , Langefeld Carl D. , Balow James , Ally Delphine S. , Kohtamaki Kimmo , Chines Peter , Birznieks Gunther , Kaleta Hong-Shi , Musick Anjene , Te Catherine , Tannenbaum Joyce , Eldridge William , Shapiro Shane , Martin Colin , Witt Alyson , So Alistair , Chang Jennie , Shurtleff Ben , Porter Rachel , Kudelko Kristina , Unni Arun , Segal Leonid , Sharaf Ravi , Blaschak-Harvan Jillian , Eriksson Johan , Tenkula Tuula , Vidgren Gabriele , Ehnholm Christian , Tuomilehto-Wolf Eva , Hagopian William , Buchanan Thomas A. , Tuomilehto Jaakko , Bergman Richard N. , Collins Francis S. , Boehnke Michael
Published: July 2002

We are conducting a genome scan at an average resolution of 10 centimorgans (cM) for type 2 diabetes susceptibility genes in 716 affected sib pairs from 477 Finnish families. To date, our best evidence for linkage is on chromosome 20 with potentially separable peaks located on both the long and short arms. The unweighted multipoint maximum logarithm of odds score (MLS) was 3.08 on 20p (location,
x̂
= 19.5 cM) under an additive model, whereas the weighted MLS was 2.06 on 20q (
x̂
= 57 cM, recurrence risk, λ̂
s
= 1.25,
P
= 0.009). Weighted logarithm of odds scores of 2.00 (
x̂
= 69.5 cM,
P
= 0.010) and 1.92 (
x̂
= 18.5 cM,
P
= 0.013) were also observed. Ordered subset analyses based on sibships with extreme mean values of diabetes-related quantitative traits yielded sets of families who contributed disproportionately to the peaks. Two-hour glucose levels in offspring of diabetic individuals gave a MLS of 2.12 (
P
= 0.0018) at 9.5 cM. Evidence from this and other studies suggests at least two diabetes-susceptibility genes on chromosome 20. We have also screened the gene for maturity-onset diabetes of the young 1, hepatic nuclear factor 4-a (
HNF-4
α) in 64 affected sibships with evidence for high chromosomal sharing at its location on chromosome 20q. We found no evidence that sequence changes in this gene accounted for the linkage results we observed.
Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex
Authors: Machesky Laura M. , Mullins R. Dyche , Higgs Henry N. , Kaiser Donald A. , Blanchoin Laurent , May Robin C. , Hall Margaret E. , Pollard Thomas D.
Published: July 2002

The Arp2/3 complex, a stable assembly of two actin-related proteins (Arp2 and Arp3) with five other subunits, caps the pointed end of actin filaments and nucleates actin polymerization with low efficiency. WASp and Scar are two similar proteins that bind the p21 subunit of the Arp2/3 complex, but their effect on the nucleation activity of the complex was not known. We report that full-length, recombinant human Scar protein, as well as N-terminally truncated Scar proteins, enhance nucleation by the Arp2/3 complex. By themselves, these proteins either have no effect or inhibit actin polymerization. The actin monomer-binding W domain and the p21-binding A domain from the C terminus of Scar are both required to activate Arp2/3 complex. A proline-rich domain in the middle of Scar enhances the activity of the W and A domains. Preincubating Scar and Arp2/3 complex with actin filaments overcomes the initial lag in polymerization, suggesting that efficient nucleation by the Arp2/3 complex requires assembly on the side of a preexisting filament—a dendritic nucleation mechanism. The Arp2/3 complex with full-length Scar, Scar containing P, W, and A domains, or Scar containing W and A domains overcomes inhibition of nucleation by the actin monomer-binding protein profilin, giving active nucleation over a low background of spontaneous nucleation. These results show that Scar and, likely, related proteins, such as the Cdc42 targets WASp and N-WASp, are endogenous activators of actin polymerization by the Arp2/3 complex.