bluuebunny/crossref_metadata_2025 huggingface dataset:
An Escherichia coli chromosomal "addiction module" regulated by guanosine [corrected] 3',5'-bispyrophosphate: a model for programmed bacterial cell death.
Authors: Aizenman E , Engelberg-Kulka H , Glaser G
Published: July 2002

"Addiction modules" consist of two genes. In most of them the product of one is long lived and toxic while the product of the second is short lived and antagonizes the toxic effect; so far, they have been described mainly in a number of prokaryotic extrachromosomal elements responsible for the postsegregational killing effect. Here we show that the chromosomal genes mazE and mazF, located in the Escherichia coli rel operon, have all of the properties required for an addiction module. Furthermore, the expression of mazEF is regulated by the cellular level of guanosine [corrected] 3',5'-bispyrophosphate, the product of the RelA protein under amino acid starvation. These properties suggest that the mazEF system may be responsible for programmed cell death in E. coli and thus may have a role in the physiology of starvation.
Use of engineered ribozymes to catalyze chimeric gene assembly.
Authors: Mikheeva S , Jarrell K A
Published: July 2002

We report the use of engineered ribozymes to shuffle exon cassettes in vitro. Specifically, we have designed derivatives of a group II intron that insert into selected sites in the human tissue plasminogen activator (t-PA) mRNA. The insertion reaction links t-PA sequences to the group II intron sequences so that trans-splicing reactions catalyzed by the intron can be employed to shuffle the t-PA sequences. We expect these results to be generalizable, so that similar ribozymes can be designed to target any desired 13 nucleotide sequence. In principle, the reactions we describe here should be able to link any RNA molecule to any other RNA molecule at any selected point.
CREB binding protein acts synergistically with steroid receptor coactivator-1 to enhance steroid receptor-dependent transcription.
Authors: Smith C L , Oñate S A , Tsai M J , O'Malley B W
Published: July 2002

Steroid receptors are ligand-regulated transcription factors that require coactivators for efficient activation of target gene expression. The binding protein of cAMP response element binding protein (CBP) appears to be a promiscuous coactivator for an increasing number of transcription factors and the ability of CBP to modulate estrogen receptor (ER)- and progesterone receptor (PR)-dependent transcription was therefore examined. Ectopic expression of CBP or the related coactivator, p300, enhanced ER transcriptional activity by up to 10-fold in a receptor- and DNA-dependent manner. Consistent with this, the 12S E1A adenoviral protein, which binds to and inactivates CBP, inhibited ER transcriptional activity, and exogenous CBP was able to partially overcome this effect. Furthermore, CBP was able to partially reverse the ability of active ER to squelch PR-dependent transcription, indicating that CBP is a common coactivator for both receptors and that CBP is limiting within these cells. To date, the only other coactivator able to significantly stimulate receptor-dependent transcription is steroid receptor coactivator-1 (SRC-1). Coexpression of CBP and SRC-1 stimulated ER and PR transcriptional activity in a synergistic manner and indicated that these two coactivators are not functional homologues. Taken together, these data suggest that both CBP and SRC-1 may function in a common pathway to efficiently activate target gene expression.
DNA sequence insertion and evolutionary variation in gene regulation.
Authors: Britten R J
Published: July 2002

Current evidence on the long-term evolutionary effect of insertion of sequence elements into gene regions is reviewed, restricted to cases where a sequence derived from a past insertion participates in the regulation of expression of a useful gene. Ten such examples in eukaryotes demonstrate that segments of repetitive DNA or mobile elements have been inserted in the past in gene regions, have been preserved, sometimes modified by selection, and now affect control of transcription of the adjacent gene. Included are only examples in which transcription control was modified by the insert. Several cases in which merely transcription initiation occurred in the insert were set aside. Two of the examples involved the long terminal repeats of mammalian endogenous retroviruses. Another two examples were control of transcription by repeated sequence inserts in sea urchin genomes. There are now six published examples in which Alu sequences were inserted long ago into human gene regions, were modified, and now are central in control/enhancement of transcription. The number of published examples of Alu sequences affecting gene control has grown threefold in the last year and is likely to continue growing. Taken together, all of these examples show that the insertion of sequence elements in the genome has been a significant source of regulatory variation in evolution.
FHIT gene alterations in head and neck squamous cell carcinomas.
Authors: Virgilio L , Shuster M , Gollin S M , Veronese M L , Ohta M , Huebner K , Croce C M
Published: July 2002

To determine whether the FHIT gene at 3p14.2 is altered in head and neck squamous cell carcinomas (HNSCC), we examined 26 HNSCC cell lines for deletions within the FHIT locus by Southern analysis, for allelic losses of specific exons FHIT by fluorescence in situ hybridization (FISH) and for integrity of FHIT transcripts. Three cell lines exhibited homozygous deletions within the FHIT gene, 55% (15/25) showed the presence of aberrant transcripts, and 65% (13/20) showed the presence of multiple cell populations with losses of different portions of FHIT alleles by FISH of FHIT genomic clones to interphase nuclei. When the data obtained by FISH and by reverse transcriptase-PCR analyses are combined, 22 of 26 cell lines showed alterations of at least one allele of the FHIT gene. Our data indicate that the FHIT gene is disrupted in HNSCCs and hence, loss of FHIT function may be important in the development and/or progression of head and neck cancers.
Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions.
Authors: Vidal M , Brachmann R K , Fattaey A , Harlow E , Boeke J D
Published: July 2002

Macromolecular interactions define many biological phenomena. Although genetic methods are available to identify novel protein-protein and DNA-protein interactions, no genetic system has thus far been described to identify molecules or mutations that dissociate known interactions. Herein, we describe genetic systems that detect such events in the yeast Saccharomyces cerevisiae. We have engineered yeast strains in which the interaction of two proteins expressed in the context of the two-hybrid system or the interaction between a DNA-binding protein and its binding site in the context of the one-hybrid system is deleterious to growth. Under these conditions, dissociation of the interaction provides a selective growth advantage, thereby facilitating detection. These methods referred to as the "reverse two-hybrid system" and "reverse one-hybrid system" facilitate the study of the structure-function relationships and regulation of protein-protein and DNA-protein interactions. They should also facilitate the selection of dissociator molecules that could be used as therapeutic agents.
Membrane fusion protein synexin (annexin VII) as a Ca2+/GTP sensor in exocytotic secretion.
Authors: Caohuy H , Srivastava M , Pollard H B
Published: July 2002

Exocytotic membrane fusion and secretion are promoted by the concerted action of GTP and Ca2+, although the precise site(s) of action in the process are not presently known. However, the calcium-dependent membrane fusion reaction driven by synexin (annexin VII) is an in vitro model for this process, which we have now found to be further activated by GTP. The mechanism of fusion activation depends on the unique ability of synexin to bind and hydrolyze GTP in a calcium-dependent manner, both in vitro and in vivo in streptolysin O-permeabilized chromaffin cells. The required [Ca2+] for GTP binding by synexin is in the range of 50-200 microM, which is known to occur at exocytotic sites in chromaffin cells, neurons, and other cell types. Previous immunolocalization studies place synexin at exocytotic sites in chromaffin cells, and we conclude that synexin is an atypical G protein that may be responsible for both detecting and mediating the Ca2+/GTP signal for exocytotic membrane fusion.
Independent regulation of JNK/p38 mitogen-activated protein kinases by metabolic oxidative stress in the liver
Authors: Mendelson Kim G. , Contois Liang-Ru , Tevosian Sergei G. , Davis Roger J. , Paulson K. Eric
Published: July 2002

The stress-activated protein kinases JNK and p38 mediate increased
gene expression and are activated by environmental stresses and
proinflammatory cytokines. Using an
in vivo
model in which
oxidative stress is generated in the liver by intracellular metabolism,
rapid protein–DNA complex formation on stress-activated AP-1 target
genes was observed. Analysis of the induced binding complexes indicates
that c-fos, c-jun, and ATF-2 were present, but also two additional jun
family members, JunB and JunD. Activation of JNK precedes increased
AP-1 DNA binding. Furthermore, JunB was shown to be a substrate for
JNK, and phosphorylation requires the N-terminal activation domain.
Unexpectedly, p38 activity was found to be constitutively active in the
liver and was down-regulated through selective dephosphorylation
following oxidative stress. One potential mechanism for p38
dephosphorylation is the rapid stress-induced activation of the
phosphatase MKP-1, which has high affinity for phosphorylated p38 as a
substrate. These data demonstrate that there are mechanisms for
independent regulation of the JNK and p38 mitogen-activated protein
kinase signal transduction pathways after metabolic oxidative stress in
the liver.
Brca2
is coordinately regulated with
Brca1
during proliferation and differentiation in mammary epithelial cells
Authors: Rajan Jayant V. , Wang Man , Marquis Sandra T. , Chodosh Lewis A.
Published: July 2002

We have analyzed the expression of the breast cancer susceptibility
gene,
Brca2
, in mammary epithelial cells as a function
of proliferation and differentiation. Our results demonstrate that
Brca2
mRNA expression is tightly regulated during
mammary epithelial proliferation and differentiation, and that this
regulation occurs coordinately with
Brca1
. Specifically,
Brca2
mRNA expression is up-regulated in rapidly
proliferating cells; is down-regulated in response to serum
deprivation; is expressed in a cell cycle-dependent manner, peaking at
the G
1
/S boundary; and is up-regulated in differentiating
mammary epithelial cells in response to glucocorticoids. In each case,
an identical pattern of expression was observed for
Brca1
. These results indicate that proliferative stimuli
modulate the mRNA expression of these two breast cancer susceptibility
genes. In addition, the coordinate regulation of
Brca1
and
Brca2
revealed by these experiments suggests that
these genes are induced by, and may function in, overlapping regulatory
pathways involved in the control of cell proliferation and
differentiation.
Mapping the universe in three dimensions
Authors: Haynes Martha P.
Published: July 2002

The determination of the three-dimensional layout of galaxies is
critical to our understanding of the evolution of galaxies and the
structures in which they lie, to our determination of the fundamental
parameters of cosmology, and to our understanding of both the past and
future histories of the universe at large. The mapping of the large
scale structure in the universe via the determination of galaxy red
shifts (Doppler shifts) is a rapidly growing industry thanks to
technological developments in detectors and spectrometers at
radio and optical wavelengths. First-order application of the red
shift-distance relation (Hubble’s law) allows the analysis of the
large-scale distribution of galaxies on scales of hundreds of
megaparsecs. Locally, the large-scale structure is very complex but the
overall topology is not yet clear. Comparison of the observed red
shifts with ones expected on the basis of other distance estimates
allows mapping of the gravitational field and the underlying total
density distribution. The next decade holds great promise for our
understanding of the character of large-scale structure and its origin.
Leptin can induce proliferation, differentiation, and functional activation of hemopoietic cells
Authors: Gainsford Timothy , Willson Tracy A. , Metcalf Donald , Handman Emanuela , McFarlane Clare , Ng Ashley , Nicola Nicos A. , Alexander Warren S. , Hilton Douglas J.
Published: July 2002

Many cytokines exert their biological effect through members of the
hemopoietin receptor family. Using degenerate oligonucleotides to the
common WSXWS motif, we have cloned from human hemopoietic cell cDNA
libraries various forms of the receptor that was recently shown to bind
the obesity hormone, leptin. mRNAs encoding long and short forms of the
human leptin receptor were found to be coexpressed in a range of human
and murine hemopoietic organs, and a subset of cells from these tissues
bound leptin at the cell surface. Ectopic expression in murine Ba/F3
and M1 cell lines revealed that the long, but not the short, form of
the leptin receptor can signal proliferation and differentiation,
respectively. In cultures of murine or human marrow cells, human leptin
exhibited no capacity to stimulate cell survival or proliferation, but
it enhanced cytokine production and phagocytosis of
Leishmania
parasites by murine peritoneal macrophages.
Our data provide evidence that, in addition to its role in fat
regulation, leptin may also be able to regulate aspects of hemopoiesis
and macrophage function.
DANA elements: a family of composite, tRNA-derived short interspersed DNA elements associated with mutational activities in zebrafish (Danio rerio).
Authors: Izsvák Z , Ivics Z , Garcia-Estefania D , Fahrenkrug S C , Hackett P B
Published: July 2002

DNA is the first SINE isolated from zebrafish (Danio rerio) exhibiting all the hallmarks of these tRNA-derived elements. DANA is unique in its clearly defined substructure of distinct cassettes. In contrast to generic SINE elements, DANA appears to have been assembled by insertions of short sequences into a progenitor, tRNA-derived element. Once associated with each other, these subunits were amplified as a new transposable element with such a remarkable success that DANA-related sequences comprise approximately 10% of the modern zebrafish genome. At least some of the sequences comprised by the full-length element were capable of movement, forming a new group of mobile, composite transposons, one of which caused an insertional mutation in the zebrafish no tail gene. Being present only in the genus Danio, and estimated to be as old as the genus itself, DANA may have played a role in Danio speciation by massive amplification and genome-wide dispersion. There are extensive DNA polymorphisms between zebrafish populations and strains detected by PCR amplification using primers specific to DANA, suggesting that the DANA element will be useful as a molecular tool for genetic and phylogenetic analyses.
Conservation of synteny between the genome of the pufferfish (Fugu rubripes) and the region on human chromosome 14 (14q24.3) associated with familial Alzheimer disease (AD3 locus)
Authors: Trower M K , Orton S M , Purvis I J , Sanseau P , Riley J , Christodoulou C , Burt D , See C G , Elgar G , Sherrington R , Rogaev E I , St George-Hyslop P , Brenner S , Dykes C W
Published: July 2002

The genome of the pufferfish (Fugu rubripes) (400 Mb) is approximately 7.5 times smaller than the human genome, but it has a similar gene repertoire to that of man. If regions of the two genomes exhibited conservation of gene order (i.e., were syntenic), it should be possible to reduce dramatically the effort required for identification of candidate genes in human disease loci by sequencing syntenic regions of the compact Fugu genome. We have demonstrated that three genes (dihydrolipoamide succinyltransferase, S31iii125, and S20i15), which are linked to FOS in the familial Alzheimer disease focus (AD3) on human chromosome 14, have homologues in the Fugu genome adjacent to Fugu cFOS. The relative gene order of cFOS, S31iii125, and S20i15 was the same in both genomes, but in Fugu these three genes lay within a 12.4-kb region, compared to >600 kb in the human AD3 locus. These results demonstrate the conservation of synteny between the genomes of Fugu and man and highlight the utility of this approach for sequence-based identification of genes in human disease loci.
Sequence scanning: A method for rapid sequence acquisition from large-fragment DNA clones.
Authors: Nurminsky D I , Hartl D L
Published: July 2002

A strategy of "sequence scanning" is proposed for rapid acquisition of sequence from clones such as bacteriophage P1 clones, cosmids, or yeast artificial chromosomes. The approach makes use of a special vector, called LambdaScan, that reliably yields subclones with inserts in the size range 8-12 kb. A number of subclones, typically 96 or 192, are chosen at random, and the ends of the inserts are sequenced using vector-specific primers. Then long-range spectrum PCR is used to order and orient the clones. This combination of shotgun and directed sequencing results in a high-resolution physical map suitable for the identification of coding regions or for comparison of sequence organization among genomes. Computer simulations indicate that, for a target clone of 100 kb, the scanning of 192 subclones with sequencing reads as short as 350 bp results in an approximate ratio of 1:2:1 of regions of double-stranded sequence, single-stranded sequence, and gaps. Longer sequencing reads tip the ratio strongly toward increased double-stranded sequence.
X-ray absorption fine structure as a monitor of zinc coordination sites during oogenesis of Xenopus laevis.
Authors: Auld D S , Falchuk K H , Zhang K , Montorzi M , Vallee B L
Published: July 2002

The x-ray absorption fine structure (XAFS) zinc K-edge steps for intact stages I,II and V,VI Xenopus laevis oocytes demonstrate that the zinc concentration is about 3 and 1 mM, respectively. However, the chi(k) function for the early stage oocytes differs markedly from that for the late one. Analysis of the XAFS data for stage I,II oocytes indicates that zinc is bound to 2.0 +/- 0.5 sulfur atoms at an average coordination distance of 2.29 +/- 0.02 angstroms and 2.0 +/- 0.5 nitrogen or oxygen (N/O) atoms at 2.02 +/- 0.02 angstroms. In marked contrast, in stage V,VI oocytes, zinc is bound to 4.1 +/- 0.4 N/O atoms at an average distance of 1.98 +/- 0.01 angstroms. Our previous studies demonstrated that 90% of the zinc in stage VI oocytes is sequestered within yolk platelets, associated with a single molecule, lipovitellin, the proteolytically processed product of vitellogenin. XAFS analysis of yolk platelets, lipovitellin, and vitellogenin demonstrates that zinc is bound to 4.0 +/- 0.5 N/O ligands at an average distance of 1.98 +/- 0.01 angstroms in each case, identical to that of stage V,VI oocytes. The higher shell contributions in the Fourier transforms indicate that two of the N/O zinc ligands are His in both stage V,VI and I,II oocytes. The results show that in stage I,II oocytes, there is a high concentration of a zinc protein whose zinc coordination site likely is composed of (His)2(Cys)2, such as, e.g., TFIIIA. As the oocytes develop, the predominant zinc species becomes one that exhibits the (His)2(N/0)2 zinc site found in lipovitellin. Hence, the ligands to the zinc atoms in intact oocytes and the changes that take place as a function of oogenesis and after their fertilization, during embryogenesis, now can be examined and explored.
What electrical measurements can say about changes in fault systems.
Authors: Madden T R , Mackie R L
Published: July 2002

Earthquake zones in the upper crust are usually more conductive than the surrounding rocks, and electrical geophysical measurements can be used to map these zones. Magnetotelluric (MT) measurements across fault zones that are parallel to the coast and not too far away can also give some important information about the lower crustal zone. This is because the long-period electric currents coming from the ocean gradually leak into the mantle, but the lower crust is usually very resistive and very little leakage takes place. If a lower crustal zone is less resistive it will be a leakage zone, and this can be seen because the MT phase will change as the ocean currents leave the upper crust. The San Andreas Fault is parallel to the ocean boundary and close enough to have a lot of extra ocean currents crossing the zone. The Loma Prieta zone, after the earthquake, showed a lot of ocean electric current leakage, suggesting that the lower crust under the fault zone was much more conductive than normal. It is hard to believe that water, which is responsible for the conductivity, had time to get into the lower crustal zone, so it was probably always there, but not well connected. If this is true, then the poorly connected water would be at a pressure close to the rock pressure, and it may play a role in modifying the fluid pressure in the upper crust fault zone. We also have telluric measurements across the San Andreas Fault near Palmdale from 1979 to 1990, and beginning in 1985 we saw changes in the telluric signals on the fault zone and east of the fault zone compared with the signals west of the fault zone. These measurements were probably seeing a better connection of the lower crust fluids taking place, and this may result in a fluid flow from the lower crust to the upper crust. This could be a factor in changing the strength of the upper crust fault zone.
Proteins associated with RNase E in a multicomponent ribonucleolytic complex.
Authors: Miczak A , Kaberdin V R , Wei C L , Lin-Chao S
Published: July 2002

The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed.
Analysis of lipopolysaccharide-response genes in B-lineage cells demonstrates that they can have differentiation stage-restricted expression and contain SH2 domains.
Authors: Kerr W G , Heller M , Herzenberg L A
Published: July 2002

Bacterial lipopolysaccharide (LPS) is a potent stimulator of B-cell activation, proliferation, and differentiation. We examined the genetic response of B-lineage cells to LPS via trapping of expressed genes with a gene-trap retrovirus. This analysis showed that expression of only a small fraction of genes is altered during LPS stimulation of B-lineage cells. Isolation of the cellular portion of the trapped LPS-response genes via 5' RACE (rapid amplification of cDNA ends) cloning identified novel genes for all the cloned loci. These novel LPS-response genes were also found to have differentiation stage-restricted expression within the B-lymphoid lineage. That LPS-response genes in B cells also have differentiation stage-restricted expression suggests that these genes may be involved in the control of B-cell function and differentiation, since the known members of this class of genes have frequently been found to play a role in the function and differentiation of B-lineage cells. The isolation of novel members of this class of genes, including a gene that contains a putative SH2 domain, will further increase our understanding of the molecular events involved in the control of B-cell differentiation and function.
Immunologic basis of transplant-associated arteriosclerosis.
Authors: Shi C , Lee W S , He Q , Zhang D , Fletcher D L , Newell J B , Haber E
Published: July 2002

Although immunosuppressive therapy minimizes the risk of graft failure due to acute rejection, transplant-associated arteriosclerosis of the coronary arteries remains a significant obstacle to the long-term survival of heart transplant recipients. The participation of specific inflammatory cell types in the genesis of this lesion was examined in a mouse model in which carotid arteries were transplanted across multiple histocompatibility barriers into seven mutant strains with immunologic defects. An acquired immune response--with the participation of CD4+ (helper) T cells, humoral antibody, and macrophages--was essential to the development of the concentric neointimal proliferation and luminal narrowing characteristic of transplant arteriosclerosis. CD8+ (cytotoxic) T cells and natural killer cells were not involved in the process. Arteries allografted into mice deficient in both T-cell receptors and humoral antibody showed almost no neointimal proliferation, whereas those grafted into mice deficient only in helper T cells, humoral antibody, or macrophages developed small neointimas. These small neointimas and the large neointimas of arteries grafted into control animals contained a similar number of inflammatory cells; however, smooth muscle cell number and collagen deposition were diminished in the small neointimas. Also, the degree of inflammatory reaction in the adventitia did not correlate with the size of the neointima. Thus, the reduction in neointimal size in arteries allografted into mice deficient in helper T cells, humoral antibody, or macrophages may be accounted for by a decrease in smooth muscle cell migration or proliferation.
Calorie restriction lowers body temperature in rhesus monkeys, consistent with a postulated anti-aging mechanism in rodents.
Authors: Lane M A , Baer D J , Rumpler W V , Weindruch R , Ingram D K , Tilmont E M , Cutler R G , Roth G S
Published: July 2002

Many studies of caloric restriction (CR) in rodents and lower animals indicate that this nutritional manipulation retards aging processes, as evidenced by increased longevity, reduced pathology, and maintenance of physiological function in a more youthful state. The anti-aging effects of CR are believed to relate, at least in part, to changes in energy metabolism. We are attempting to determine whether similar effects occur in response to CR in nonhuman primates. Core (rectal) body temperature decreased progressively with age from 2 to 30 years in rhesus monkeys fed ad lib (controls) and is reduced by approximately 0.5 degrees C in age-matched monkeys subjected to 6 years of a 30% reduction in caloric intake. A short-term (1 month) 30% restriction of 2.5-year-old monkeys lowered subcutaneous body temperature by 1.0 degrees C. Indirect calorimetry showed that 24-hr energy expenditure was reduced by approximately 24% during short-term CR. The temporal association between reduced body temperature and energy expenditure suggests that reductions in body temperature relate to the induction of an energy conservation mechanism during CR. These reductions in body temperature and energy expenditure are consistent with findings in rodent studies in which aging rate was retarded by CR, now strengthening the possibility that CR may exert beneficial effects in primates analogous to those observed in rodents.
Phosphorylation of the fused protein kinase in response to signaling from hedgehog.
Authors: Thérond P P , Knight J D , Kornberg T B , Bishop J M
Published: July 2002

The hedgehog gene (hh) of Drosophila melanogaster exerts both short- and long-range effects on cell patterning during development. The product of hedgehog is a secreted protein that apparently acts by triggering an intra-cellular signaling pathway, but little is known about the details of that pathway. The Drosophila gene fused (fu) encodes a serine/threonine-protein kinase that genetic experiments have implicated in signaling initiated by hedgehog. Here we report that the fused protein is phosphorylated during the course of Drosophila embryogenesis, as a result of hedgehog activity. In cell culture, phosphorylation of fused protein occurs in response to the biologically active form of hedgehog and cannot be blocked by activation of protein kinase A, which is thought to be an antagonist of signaling from hedgehog. These results suggest that fused and protein kinase A function downstream of hedgehog but in parallel pathways that eventually converge distal to fused. The reconstruction of signaling from hedgehog in cell culture should provide further access to the mechanisms by which hedgehog acts.
Direct conversion of an oligopeptide from a β-sheet to an α-helix: A model for amyloid formation
Authors: Zhang Shuguang , Rich Alexander
Published: July 2002

A 16-amino acid oligopeptide forms a stable β-sheet structure in
water. In physiological solutions it is able to self-assemble to form a
macroscopic matrix that stains with Congo red. On raising the
temperature of the aqueous solution above 70°C, an abrupt structural
transition occurs in the CD spectra from a β-sheet to a stable
α-helix without a detectable random-coil intermediate. With cooling,
it retained the α-helical form and took several weeks at room
temperature to partially return to the β-sheet form. Slow formation
of the stable β-sheet structure thus shows kinetic irreversibility.
Such a formation of very stable β-sheet structures is found in the
amyloid of a number of neurological diseases. This oligopeptide could
be a model system for studying the protein conformational changes that
occurs in scrapie or Alzheimer disease. The abrupt and direct
conversion from a β-sheet to an α-helix may also be found in other
processes, such as protein folding and protein–protein interaction.
Furthermore, such drastic structure changes may also be exploited in
biomaterials designed as sensors to detect environmental changes.
The emergence of form by replication
Authors: Velarde M. G. , Nekorkin V. I. , Kazantsev V. B. , Ross J.
Published: July 2002

It is shown with a simple mathematical model that if a system exhibits a given form (a spatial structure) and is put in contact with another system of the same type but in a state of spatial disorder, then under certain conditions their mutual interaction as they evolve in time allows replication of form in the disordered system with a controllable degree of faithfulness.
Activation of the orphan receptor RIP14 by retinoids
Authors: Zavacki Ann Marie , Lehmann Jürgen M. , Seol Wongi , Willson Timothy M. , Kliewer Steven A. , Moore David D.
Published: July 2002

Retinoids are crucial regulators of a wide variety of processes in both developing and adult animals. These effects are thought to be mediated by the retinoic acid (RA) receptors and the retinoid X receptors (RXRs). We have identified an additional retinoid-activated receptor that is neither a retinoic acid receptors nor an RXR. RXR-interacting protein 14 (RIP14), a recently described orphan member of the nuclear receptor superfamily, can be activated by either all-
trans
-RA (tRA) or the synthetic retinoid TTNPB {[
E
]-4-[2-(5, 6, 7, 8-tetrahydro-5, 5, 8, 8-tetramethyl-2-naphthalenyl)propen-1-yl]benzoic acid}. RIP14 binds to DNA as a heterodimer with RXR. In the presence of either tRA or TTNPB, the addition of 9-
cis
-RA or the RXR-specific agonist LG1069 {4-[1-(3, 5, 5, 8, 8-pentamethyl-5, 6, 7, 8-tertrahydro-2-naphthyl)ethenyl]benzoic acid} results in additional activation. Mutations of the ligand-dependent transcriptional activation functions indicate that TTNPB activates the RIP14 component of the RIP14–RXR heterodimer, that 9-
cis
-RA and LG1069 activate RXR, and that tRA activates via both RIP14 and RXR. Despite the very effective activation of RIP14 by tRA or TTNPB, relatively high concentrations of these compounds are required, and no evidence for direct binding of either compound was obtained using several approaches. These results suggest that RIP14 is the receptor for an as-yet-unidentified retinoid metabolite.
The SH3p4/Sh3p8/SH3p13 protein family: Binding partners for synaptojanin and dynamin via a Grb2-like Src homology 3 domain
Authors: Ringstad Niels , Nemoto Yasuo , De Camilli Pietro
Published: July 2002

The GTPase dynamin I and the inositol 5-phosphatase synaptojanin are nerve terminal proteins implicated in synaptic vesicle recycling. Both proteins contain COOH-terminal proline-rich domains that can interact with a variety of Src homology 3 (SH3) domains. A major physiological binding partner for dynamin I and synaptojanin in the nervous system is amphiphysin I, an SH3 domain-containing protein also concentrated in nerve terminals. We have used the proline-rich tail of synaptojanin to screen a rat brain library by the two-hybrid method to identify additional interacting partners of synaptojanin. Three related proteins containing SH3 domains that are closely related to the SH3 domains of Grb2 were isolated: SH3p4, SH3p8, and SH3p13. Further biochemical studies demonstrated that the SH3p4/8/13 proteins bind to both synaptojanin and dynamin I. The SH3p4/8/13 transcripts are differentially expressed in tissues: SH3p4 mRNA was detected only in brain, SH3p13 mRNA was present in brain and testis, and the SH3p8 transcript was detected at similar levels in multiple tissues. Members of the SH3p4/8/13 protein family were found to be concentrated in nerve terminals, and pools of synaptojanin and dynamin I were coprecipitated from brain extracts with antibodies recognizing SH3p4/8/13. These findings underscore the important role of SH3-mediated interactions in synaptic vesicle recycling.
Peropsin, a novel visual pigment-like protein located in the apical microvilli of the retinal pigment epithelium
Authors: Sun Hui , Gilbert Debra J. , Copeland Neal G. , Jenkins Nancy A. , Nathans Jeremy
Published: July 2002

A visual pigment-like protein, referred to as peropsin, has been identified by large-scale sequencing of cDNAs derived from human ocular tissues. The corresponding mRNA was found only in the eye, where it is localized to the retinal pigment epithelium (RPE). Peropsin immunoreactivity, visualized by light and electron microscopy, localizes the protein to the apical face of the RPE, and most prominently to the microvilli that surround the photoreceptor outer segments. These observations suggest that peropsin may play a role in RPE physiology either by detecting light directly or by monitoring the concentration of retinoids or other photoreceptor-derived compounds.
Isolation of quelling-defective (
qde
) mutants impaired in posttranscriptional transgene-induced gene silencing in
Neurospora crassa
Authors: Cogoni Carlo , Macino Giuseppe
Published: July 2002

We report the isolation of 15
Neurospora crassa
mutants defective in “quelling” or transgene-induced gene silencing. These quelling-defective mutants (
qde
) belonging to three complementation groups have provided insights into the mechanism of posttranscriptional gene silencing in
N. crassa
. The recessive nature of the
qde
mutations indicates that the encoded gene products act in trans. We show that when
qde
genes are mutated in a transgenic-induced silenced strain containing many copies of the transgene, the expression of the endogenous gene is maintained despite the presence of transgene sense RNA, the molecule proposed to trigger quelling. Moreover, the
qde
mutants failed to show quelling when tested with another gene, suggesting that they may be universally defective in transgene-induced gene silencing. As such,
qde
genes may be involved in sensing aberrant sense RNA and/or targeting/degrading the native mRNA. The
qde
mutations may be used to isolate the genes encoding the first components of the quelling mechanism. Moreover, these quelling mutants may be important in applied and basic research for the creation of strains able to overexpress a transgene.
RGS4 and GAIP are GTPase-activating proteins for G
qα
and block activation of phospholipase Cβ by γ-thio-GTP-G
qα
Authors: Hepler John R. , Berman David M. , Gilman Alfred G. , Kozasa Tohru
Published: July 2002

RGS proteins constitute a newly appreciated and large group of negative
r
egulators of
G
protein
s
ignaling. Four members of the RGS family act as GTPase-activating proteins (GAPs) with apparent specificity for members of the G
iα
subfamily of G protein subunits. We demonstrate here that two RGS proteins, RGS4 and GAIP, also act as GAPs for G
qα
, the G
α
protein responsible for activation of phospholipase Cβ. Furthermore, these RGS proteins block activation of phospholipase Cβ by guanosine 5′-(3-
O
-thio)triphosphate-G
qα
. GAP activity does not explain this effect, which apparently results from occlusion of the binding site on G
α
for effector. Inhibitory effects of RGS proteins on G protein-mediated signaling pathways can be demonstrated by simple mixture of RGS4 or GAIP with plasma membranes.
Intron “sliding” and the diversity of intron positions
Authors: Stoltzfus Arlin , Logsdon John M. , Palmer Jeffrey D. , Doolittle W. Ford
Published: July 2002

Alignments of homologous genes typically reveal a great diversity of intron locations, far more than could fit comfortably in a single gene. Thus, a minority of these intron positions could be inherited from a single ancestral gene, but the larger share must be attributed to subsequent events of intron gain or intron “sliding” (movement from one position to another within a gene). Intron sliding has been argued from cases of discordant introns and from putative spatial clustering of intron positions. A list of 32 cases of discordant introns is presented here. Most of these cases are found to be artefactual. The spatial and phylogenetic distributions of intron positions from five published compilations of gene data, comprising 205 intron positions, have been examined systematically for evidence of intron sliding. The results suggest that sliding, if it occurs at all, has contributed little to the diversity of intron positions.
Efficient allelic exchange and transposon mutagenesis in
Mycobacterium tuberculosis
Authors: Pelicic Vladimir , Jackson Mary , Reyrat Jean-Marc , Jacobs William R. , Gicquel Brigitte , Guilhot Christophe
Published: July 2002

A better understanding of
Mycobacterium tuberculosis
virulence mechanisms is highly dependent on the design of efficient mutagenesis systems. A system enabling the positive selection of insertional mutants having lost the delivery vector was developed. It uses ts-
sacB
vectors, which combine the counterselective properties of the
sacB
gene and a mycobacterial thermosensitive origin of replication and can therefore be efficiently counterselected on sucrose at 39°C. This methodology allowed the construction of
M. tuberculosis
transposition mutant libraries. Greater than 10
6
mutants were obtained, far exceeding the number theoretically required to obtain at least one insertion in every nonessential gene. This system is also efficient for gene exchange mutagenesis as demonstrated with the
purC
gene: 100% of the selected clones were allelic exchange mutants. Therefore, a single, simple methodology has enabled us to develop powerful mutagenesis systems, the lack of which was a major obstacle to the genetic characterization of
M. tuberculosis
.
Massive targeting of liposomes, surface-modified with anionized albumins, to hepatic endothelial cells
Authors: Kamps Jan A. A. M. , Morselt Henriëtte W. M. , Swart Pieter J. , Meijer Dick K. F. , Scherphof Gerrit L.
Published: July 2002

Human serum albumin (HSA) derivatized with
cis
-aconitic anhydride was covalently coupled to liposomes with a size of approximately 100 nm [polyaconitylated HSA (Aco-HSA) liposomes]. Within 30 min after injection into a rat, Aco-HSA liposomes were completely cleared from the blood and almost exclusively taken up by the liver, whereas in control liposomes 80% was still present in the blood at that time. Endothelial cells were shown to account for almost two-thirds of the hepatic uptake of the Aco-HSA liposomes, the remainder being recovered mainly in the liver macrophages (Kupffer cells). With fluorescently labeled liposomes it was shown that the Aco-HSA liposomes target a vast majority (>85%) of the cells in the endothelial cell population. Control liposomes were not taken up to a significant extent by the endothelial cells. Uptake of Aco-HSA liposomes by both endothelial and Kupffer cells was inhibited by preinjection with polyinosinic acid, indicating the involvement of scavenger receptors in the uptake process. The uptake of Aco-HSA liposomes by liver endothelial cells was dependent on liposome size; with increasing liposome diameter endothelial cell uptake decreased in favor of Kupffer cell uptake. We have demonstrated that massive
in vivo
targeting of liposomes to a defined cell population other than macrophages is possible. Aco-HSA liposomes thus may represent an attractive drug carrier system for treatment of various liver or liver endothelium-associated disorders.
Yersinia enterocolitica
induces apoptosis in macrophages by a process requiring functional type III secretion and translocation mechanisms and involving YopP, presumably acting as an effector protein
Authors: Mills Scott D. , Boland Anne , Sory Marie-Paule , van der Smissen Patrick , Kerbourch Corinne , Finlay B. Brett , Cornelis Guy R.
Published: July 2002

Yersiniae
, causative agents of plague and gastrointestinal diseases, secrete and translocate Yop effector proteins into the cytosol of macrophages, leading to disruption of host defense mechanisms. It is shown in this report that
Yersinia enterocolitica
induces apoptosis in macrophages and that this effect depends on YopP. Functional secretion and translocation mechanisms are required for YopP to act, strongly suggesting that this protein exerts its effect intracellularly, after translocation into the macrophages. YopP shows a high level of sequence similarity with AvrRxv, an avirulence protein from
Xanthomonas campestris
, a plant pathogen that induces programmed cell death in plant cells. This indicates possible similarities between the strategies used by pathogenic bacteria to elicit programmed cell death in both plant and animal hosts.
Yeast microarrays for genome wide parallel genetic and gene expression analysis
Authors: Lashkari Deval A. , DeRisi Joseph L. , McCusker John H. , Namath Allen F. , Gentile Cristl , Hwang Seung Y. , Brown Patrick O. , Davis Ronald W.
Published: July 2002

We have developed high-density DNA microarrays of yeast ORFs. These microarrays can monitor hybridization to ORFs for applications such as quantitative differential gene expression analysis and screening for sequence polymorphisms. Automated scripts retrieved sequence information from public databases to locate predicted ORFs and select appropriate primers for amplification. The primers were used to amplify yeast ORFs in 96-well plates, and the resulting products were arrayed using an automated micro arraying device. Arrays containing up to 2,479 yeast ORFs were printed on a single slide. The hybridization of fluorescently labeled samples to the array were detected and quantitated with a laser confocal scanning microscope. Applications of the microarrays are shown for genetic and gene expression analysis at the whole genome level.
Influenza hemagglutinin is spring-loaded by a metastable native conformation
Authors: Carr Chavela M. , Chaudhry Charu , Kim Peter S.
Published: July 2002

Enveloped viruses enter cells by protein-mediated membrane fusion. For influenza virus, membrane fusion is regulated by the conformational state of the hemagglutinin (HA) protein, which switches from a native (nonfusogenic) structure to a fusion-active (fusogenic) conformation when exposed to the acidic environment of the cellular endosome. Here we demonstrate that destabilization of HA at neutral pH, with either heat or the denaturant urea, triggers a conformational change that is biochemically indistinguishable from the change triggered by low pH. In each case, the conformational change is coincident with induction of membrane-fusion activity, providing strong evidence that the fusogenic structure is formed. These results indicate that the native structure of HA is trapped in a metastable state and that the fusogenic conformation is released by destabilization of native structure. This strategy may be shared by other enveloped viruses, including those that enter the cell at neutral pH, and could have implications for understanding the membrane-fusion step of HIV infection.
The anticoagulant activation of antithrombin by heparin
Authors: Jin Lei , Abrahams Jan Pieter , Skinner Richard , Petitou Maurice , Pike Robert N. , Carrell Robin W.
Published: July 2002

Antithrombin, a plasma serpin, is relatively inactive as an inhibitor of the coagulation proteases until it binds to the heparan side chains that line the microvasculature. The binding specifically occurs to a core pentasaccharide present both in the heparans and in their therapeutic derivative heparin. The accompanying conformational change of antithrombin is revealed in a 2.9-Å structure of a dimer of latent and active antithrombins, each in complex with the high-affinity pentasaccharide. Inhibitory activation results from a shift in the main sheet of the molecule from a partially six-stranded to a five-stranded form, with extrusion of the reactive center loop to give a more exposed orientation. There is a tilting and elongation of helix D with the formation of a 2-turn helix P between the C and D helices. Concomitant conformational changes at the heparin binding site explain both the initial tight binding of antithrombin to the heparans and the subsequent release of the antithrombin–protease complex into the circulation. The pentasaccharide binds by hydrogen bonding of its sulfates and carboxylates to Arg-129 and Lys-125 in the D-helix, to Arg-46 and Arg-47 in the A-helix, to Lys-114 and Glu-113 in the P-helix, and to Lys-11 and Arg-13 in a cleft formed by the amino terminus. This clear definition of the binding site will provide a structural basis for developing heparin analogues that are more specific toward their intended target antithrombin and therefore less likely to exhibit side effects.