bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of hydrogen on shallow dopants in crystalline silicon
Authors: Pantelides Sokrates T.
Published: July 2002

Passivation of shallow impurities by H has been attributed to H-impurity pairing in both p-type and n-type Si. We show that existing interpretations of data were based on contradictory assumptions and that a coherent interpretation of all the data can only be obtained if one assumes that diffusing H has a donor level in the gap. A novel interpretation emerges: In p-type material, passivation is due to direct compensation, so that pairing is a consequence, not a cause of passivation; in n-type material, passivation is indeed due to pairing, but is suppressed by H2 formation and possibly other reactions. Several predictions are made and new experiments are proposed as tests.
Estimation of the thickness of thin metal sheet using laser generated ultrasound
Authors: Dewhurst R. J. , Edwards C. , McKie A. D. W. , Palmer S. B.
Published: July 2002

A noncontacting technique has been developed to measure the thickness of thin metal sheet by using a pulsed laser to generate both symmetric and antisymmetric Lamb waves. These have been detected with a laser interferometer. Analysis of the waveforms allows an estimation of the sheet thickness and accuracies to within 2% are attainable on sheets as thin as 27 μm.
Oxidation of silicon by a low-energy ion beam: Experiment and model
Authors: Todorov S. S. , Fossum E. R.
Published: July 2002

The self-limiting oxidation of silicon by a low-energy ion beam (40–120 eV) is described by an implantation-sputtering model. The thin oxide (40–50 Å) is grown primarily by a surface implantation process which leads to a logarithmic increase of oxide thickness with dose in the absence of sputtering. At higher energies (100 eV), the sputtering of the growing film leads to net self-limiting growth. The model, which does not include adjustable parameters, is used to describe the dose evolution of the oxide growth as a function of beam energy. The implantation-sputtering model is found to be in excellent agreement with experimental observations.
Ca
2+
-desensitizing effect of a deletion mutation ΔK210 in cardiac troponin T that causes familial dilated cardiomyopathy
Authors: Morimoto S. , Lu Q.-W. , Harada K. , Takahashi-Yanaga F. , Minakami R. , Ohta M. , Sasaguri T. , Ohtsuki I.
Published: July 2002

A deletion mutation ΔK210 in cardiac troponin T (cTnT) was recently found to cause familial dilated cardiomyopathy (DCM). To explore the effect of this mutation on cardiac muscle contraction under physiological conditions, we determined the Ca
2+
-activated force generation in permeabilized rabbit cardiac muscle fibers into which the mutant and wild-type cTnTs were incorporated by using our TnT exchange technique. The free Ca
2+
concentrations required for the force generation were higher in the mutant cTnT-exchanged fibers than in the wild-type cTnT-exchanged ones, with no statistically significant differences in maximal force-generating capability and cooperativity. Exchanging the mutant cTnT into isolated cardiac myofibrils also increased the free Ca
2+
concentrations required for the activation of ATPase. In contrast, a deletion mutation ΔE160 in cTnT that causes familial hypertrophic cardiomyopathy (HCM) decreased the free Ca
2+
concentrations required for force generation, just as in the case of the other HCM-causing mutations in cTnT. The results indicate that cTnT mutations found in the two distinct forms of cardiomyopathy (i.e., HCM and DCM) change the Ca
2+
sensitivity of cardiac muscle contraction in opposite directions. The present study strongly suggests that Ca
2+
desensitization of force generation in sarcomere is a primary mechanism for the pathogenesis of DCM associated with the deletion mutation ΔK210 in cTnT.
Adaptation of the bovine spongiform encephalopathy agent to primates and comparison with Creutzfeldt– Jakob disease: Implications for human health
Authors: Lasmézas Corinne Ida , Fournier Jean-Guy , Nouvel Virginie , Boe Hermann , Marcé Domíníque , Lamoury François , Kopp Nicolas , Hauw Jean-Jacques , Ironside James , Bruce Moira , Dormont Dominique , Deslys Jean-Philippe
Published: July 2002

There is substantial scientific evidence to support the
notion that bovine spongiform encephalopathy (BSE) has contaminated
human beings, causing variant Creutzfeldt–Jakob disease (vCJD). This
disease has raised concerns about the possibility of an iatrogenic
secondary transmission to humans, because the biological properties of
the primate-adapted BSE agent are unknown. We show that
(
i
) BSE can be transmitted from primate to primate by
intravenous route in 25 months, and (
ii
) an iatrogenic
transmission of vCJD to humans could be readily recognized
pathologically, whether it occurs by the central or peripheral route.
Strain typing in mice demonstrates that the BSE agent adapts to
macaques in the same way as it does to humans and confirms that the BSE
agent is responsible for vCJD not only in the United Kingdom but also
in France. The agent responsible for French iatrogenic growth
hormone-linked CJD taken as a control is very different from vCJD but
is similar to that found in one case of sporadic CJD and one sheep
scrapie isolate. These data will be key in identifying the origin of
human cases of prion disease, including accidental vCJD transmission,
and could provide bases for vCJD risk assessment.
Point mutant mice with hypersensitive α4 nicotinic receptors show dopaminergic deficits and increased anxiety
Authors: Labarca Cesar , Schwarz Johannes , Deshpande Purnima , Schwarz Sigrid , Nowak Mark W. , Fonck Carlos , Nashmi Raad , Kofuji Paulo , Dang Hong , Shi Wenmei , Fidan Melihat , Khakh Baljit S. , Chen Zhoufeng , Bowers Barbara J. , Boulter Jim , Wehner Jeanne M. , Lester Henry A.
Published: July 2002

Knock-in mice were generated that harbored a leucine-to-serine mutation in the α4 nicotinic receptor near the gate in the channel pore. Mice with intact expression of this hypersensitive receptor display dominant neonatal lethality. These mice have a severe deficit of dopaminergic neurons in the substantia nigra, possibly because the hypersensitive receptors are continuously activated by normal extracellular choline concentrations. A strain that retains the neo selection cassette in an intron has reduced expression of the hypersensitive receptor and is viable and fertile. The viable mice display increased anxiety, poor motor learning, excessive ambulation that is eliminated by very low levels of nicotine, and a reduction of nigrostriatal dopaminergic function upon aging. These knock-in mice provide useful insights into the pathophysiology of sustained nicotinic receptor activation and may provide a model for Parkinson's disease.
Inhibition of cyclooxygenase 2 blocks human cytomegalovirus replication
Authors: Zhu Hua , Cong Jian-Ping , Yu Deborah , Bresnahan Wade A. , Shenk Thomas E.
Published: July 2002

Cyclooxygenase 2 (COX-2) mRNA, protein, and activity are transiently induced after infection of human fibroblasts with human cytomegalovirus. Prostaglandin E
2
, the product of COX-2 activity, is transiently increased by a factor of >50 in cultures of virus-infected fibroblasts. Both specific (BMS-279652, 279654, and 279655) and nonspecific (indomethacin) COX-2 inhibitors can abrogate the virus-mediated induction of prostaglandin E
2
accumulation. Levels of COX-2 inhibitors that completely block the induction of COX-2 activity, but do not compromise cell viability, reduce the yield of human cytomegalovirus in human fibroblasts by a factor of >100. Importantly, the yield of infectious virus can be substantially restored by the addition of prostaglandin E
2
together with the inhibitory drug. This finding argues that elevated levels of prostaglandin E
2
are required for efficient replication of human cytomegalovirus in fibroblasts. COX-2 inhibitors block the accumulation of immediate-early 2 mRNA and protein, but have little effect on the levels of immediate-early 1 mRNA and protein. Viral DNA replication and the accumulation of some, but not all, early and late mRNAs are substantially blocked by COX-2 inhibitors. Elevated levels of prostaglandin E
2
apparently facilitate the production of immediate-early 2 protein. The failure to produce normal levels of this critical viral regulatory protein in the presence of COX-2 inhibitors might block normal progression beyond the immediate-early phase of human cytomegalovirus infection.
Declining brain activity in cognitively normal apolipoprotein E ɛ4 heterozygotes: A foundation for using positron emission tomography to efficiently test treatments to prevent Alzheimer's disease
Authors: Reiman Eric M. , Caselli Richard J. , Chen Kewei , Alexander Gene E. , Bandy Daniel , Frost Jennifer
Published: July 2002

Cross-sectional positron emission tomography (PET) studies find
that cognitively normal carriers of the apolipoprotein E (APOE) ɛ4
allele, a common Alzheimer's susceptibility gene, have abnormally low
measurements of the cerebral metabolic rate for glucose (CMRgl) in the
same regions as patients with Alzheimer's dementia. In this article,
we characterize longitudinal CMRgl declines in cognitively normal ɛ4
heterozygotes, estimate the power of PET to test the efficacy of
treatments to attenuate these declines in 2 years, and consider how
this paradigm could be used to efficiently test the potential of
candidate therapies for the prevention of Alzheimer's disease. We
studied 10 cognitively normal ɛ4 heterozygotes and 15 ɛ4
noncarriers 50–63 years of age with a reported family history of
Alzheimer's dementia before and after an interval of approximately 2
years. The ɛ4 heterozygotes had significant CMRgl declines in the
vicinity of temporal, posterior cingulate, and prefrontal cortex, basal
forebrain, parahippocampal gyrus, and thalamus, and these declines were
significantly greater than those in the ɛ4 noncarriers. In testing
candidate primary prevention therapies, we estimate that between 50 and
115 cognitively normal ɛ4 heterozygotes are needed per active and
placebo treatment group to detect a 25% attenuation in these CMRgl
declines with 80% power and
P
= 0.005 in 2 years.
Assuming these CMRgl declines are related to the predisposition to
Alzheimer's dementia, this study provides a paradigm for testing the
potential of treatments to prevent the disorder without having to
study thousands of research subjects or wait many years to
determine whether or when treated individuals develop symptoms.
Identification with a recombinant antibody of an inner-ear cytokeratin, a marker for hair-cell differentiation
Authors: Cyr Janet L. , Bell Andrea M. , Hudspeth A. J.
Published: July 2002

Extensive biochemical characterization of cells in the inner ear has been hampered by a lack of tools with which to identify inner-ear proteins. By using a single-chain antibody fragment isolated from a bacteriophage-displayed library, we have identified a cytokeratin that is abundant in nonsensory cells of the frog inner ear. Although the progenitors of hair cells exhibit strong immunoreactivity to this cytokeratin, the signal declines in immature hair cells and vanishes as the cells mature. The correlation between diminished immunoreactivity and hair-cell differentiation indicates that the cytokeratin is down-regulated during the transition from a nonsensory to a sensory cell and suggests that the marker is an early index of hair-cell differentiation.
Oligomerization and activation of caspase-9, induced by Apaf-1 CARD
Authors: Shiozaki Eric N. , Chai Jijie , Shi Yigong
Published: July 2002

Apaf-1 facilitates the proteolytic activation of procaspase-9 and maintains the hyperactive state of the processed caspase-9. The underlying molecular mechanisms for these activities remain poorly characterized. Here we report that the isolated Apaf-1 caspase recruitment domain (CARD) forms a large hetero-oligomer with the active caspase-9. The catalytic activity of caspase-9 is significantly enhanced in this complex, demonstrating that Apaf-1 CARD allosterically up-regulates caspase-9 activity. Point mutations that inactivate the interactions between Apaf-1 CARD and the prodomain of caspase-9 also abolished the formation of this complex. Based on these observations, we discuss the implications of this complex on the observed Apaf-1 function.
Identification and characterization of a host protein required for efficient template selection in viral RNA replication
Authors: Díez Juana , Ishikawa Masayuki , Kaido Masanori , Ahlquist Paul
Published: July 2002

Biochemical studies suggest that positive-strand RNA virus replication involves host as well as viral functions. Brome mosaic virus (BMV) is a member of the alphavirus-like superfamily of animal and plant positive-strand RNA viruses. Yeast expressing the BMV RNA replication proteins 1a and 2a supports BMV RNA replication and mRNA synthesis. Using the ability of BMV to replicate in yeast, we show that efficient BMV RNA replication requires Lsm1p, a yeast protein related to core RNA splicing factors but shown herein to be cytoplasmic. Haploid yeast with an Lsm1p mutation was defective in an early template selection step in BMV RNA replication, involving the helicase-like replication protein 1a and an internal viral RNA element conserved with tRNAs. Lsm1p dependence of this interaction was suppressed by adding 3′ poly(A) to the normally unpolyadenylated BMV RNA. Our results show Lsm1p involvement in a specific step of BMV RNA replication and connections between Lsm1p and poly(A) function, possibly through interaction with factors binding mRNA 5′ ends.
Adeno-associated virus site-specifically integrates into a muscle-specific DNA region
Authors: Dutheil Nathalie , Shi Fabiao , Dupressoir Thierry , Linden R. Michael
Published: July 2002

The nonpathogenic human virus adeno-associated virus type 2 (AAV) has evolved the potentially unique strategy to establish latency by site-specifically integrating its genome into human chromosome 19 (19q13.3-qter) at a locus designated
AAVS1
. This nonhomologous, site-specific recombination of viral DNA with the human genome provides a basis for developing targeted gene therapy vectors. To assess whether the region surrounding
AAVS1
might have contributed to the selection of the specific integration site, we have investigated this locus. Here, we show that
AAVS1
is closely linked to the slow skeletal troponin T gene,
TNNT1
, which has been mapped previously to 19q13.4. In support of this idea, we demonstrate that site-specific AAV DNA integration can result in the formation of
TNNT1-
AAV junctions. The question now arises whether muscle represents a natural target tissue for latent AAV infection. This possibility is of additional interest in view of recent observations that muscle tissue is particularly well suited for AAV-mediated gene transfer. The question also occurs whether latent infection by AAV can lead to phenotypic changes of the multinucleated muscle fiber cells.
Slowed conduction and ventricular tachycardia after targeted disruption of the cardiac sodium channel gene
Scn5a
Authors: Papadatos G. Alex , Wallerstein Polly M. R. , Head Catherine E. G. , Ratcliff Rosemary , Brady Peter A. , Benndorf Klaus , Saumarez Richard C. , Trezise Ann E. O. , Huang Christopher L.-H. , Vandenberg Jamie I. , Colledge William H. , Grace Andrew A.
Published: July 2002

Voltage-gated sodium channels drive the initial depolarization phase of the cardiac action potential and therefore critically determine conduction of excitation through the heart. In patients, deletions or loss-of-function mutations of the cardiac sodium channel gene,
SCN5A
, have been associated with a wide range of arrhythmias including bradycardia (heart rate slowing), atrioventricular conduction delay, and ventricular fibrillation. The pathophysiological basis of these clinical conditions is unresolved. Here we show that disruption of the mouse cardiac sodium channel gene,
Scn5a
, causes intrauterine lethality in homozygotes with severe defects in ventricular morphogenesis whereas heterozygotes show normal survival. Whole-cell patch clamp analyses of isolated ventricular myocytes from adult
Scn5a
+/−
mice demonstrate a ≈50% reduction in sodium conductance.
Scn5a
+/−
hearts have several defects including impaired atrioventricular conduction, delayed intramyocardial conduction, increased ventricular refractoriness, and ventricular tachycardia with characteristics of reentrant excitation. These findings reconcile reduced activity of the cardiac sodium channel leading to slowed conduction with several apparently diverse clinical phenotypes, providing a model for the detailed analysis of the pathophysiology of arrhythmias.
Inclusion body myositis-like phenotype induced by transgenic overexpression of βAPP in skeletal muscle
Authors: Sugarman Michael C. , Yamasaki Tritia R. , Oddo Salvatore , Echegoyen Julio C. , Murphy M. Paul , Golde Todd E. , Jannatipour Mehrdad , Leissring Malcolm A. , LaFerla Frank M.
Published: July 2002

Inclusion body myositis (IBM), the most common age-related muscle disease in the elderly population, is an incurable disorder leading to severe disability. Sporadic IBM has an unknown etiology, although affected muscle fibers are characterized by many of the pathobiochemical alterations traditionally associated with neurodegenerative brain disorders such as Alzheimer's disease. Accumulation of the amyloid-β peptide, which is derived from proteolysis of the larger amyloid-β precursor protein (βAPP), seems to be an early pathological event in Alzheimer's disease and also in IBM, where in the latter, it predominantly occurs intracellularly within affected myofibers. To elucidate the possible role of βAPP mismetabolism in the pathogenesis of IBM, transgenic mice were derived in which we selectively targeted βAPP overexpression to skeletal muscle by using the muscle creatine kinase promoter. Here we report that older (>10 months) transgenic mice exhibit intracellular immunoreactivity to βAPP and its proteolytic derivatives in skeletal muscle. In this transgenic model, selective overexpression of βAPP leads to the development of a subset of other histopathological and clinical features characteristic of IBM, including centric nuclei, inflammation, and deficiencies in motor performance. These results are consistent with a pathogenic role for βAPP mismetabolism in human IBM.
Dynamics of Fos-Jun-NFAT1 complexes
Authors: Ramirez-Carrozzi Vladimir R. , Kerppola Tom K.
Published: July 2002

Transcription initiation in eukaryotes is controlled by
nucleoprotein complexes formed through cooperative interactions among
multiple transcription regulatory proteins. These complexes may be
assembled via stochastic collisions or defined pathways. We
investigated the dynamics of Fos-Jun-NFAT1 complexes by using a
multicolor fluorescence resonance energy transfer assay. Fos-Jun
heterodimers can bind to AP-1 sites in two opposite orientations, only
one of which is populated in mature Fos-Jun-NFAT1 complexes. We studied
the reversal of Fos-Jun binding orientation in response to NFAT1 by
measuring the efficiencies of energy transfer from donor fluorophores
linked to opposite ends of an oligonucleotide to an acceptor
fluorophore linked to one subunit of the heterodimer. The reorientation
of Fos-Jun by NFAT1 was not inhibited by competitor oligonucleotides or
heterodimers. The rate of Fos-Jun reorientation was faster than the
rate of heterodimer dissociation at some binding sites. The facilitated
reorientation of Fos-Jun heterodimers therefore can enhance the
efficiency of Fos-Jun-NFAT1 complex formation. We also examined the
influence of the preferred orientation of Fos-Jun binding on the
stability and transcriptional activity of Fos-Jun-NFAT1 complexes.
Complexes formed at sites where Fos-Jun favored the same binding
orientation in the presence and absence of NFAT1 exhibited an 8-fold
slower dissociation rate than complexes formed at sites where Fos-Jun
favored the opposite binding orientation. Fos-Jun-NFAT1 complexes also
exhibited greater transcription activation at promoter elements that
favored the same orientation of Fos-Jun binding in the presence and
absence of NFAT1. Thus, the orientation of heterodimer binding can
influence both the dynamics and promoter selectivity of
multiprotein transcription regulatory complexes.
The yeast A kinases differentially regulate iron uptake and respiratory function
Authors: Robertson Laura S. , Causton Helen C. , Young Richard A. , Fink Gerald R.
Published: July 2002

Yeast has three A kinase catalytic subunits, which have greater than 75% identity and are encoded by the
TPK
genes (
TPK1
,
TPK2
, and
TPK3
) [Toda, T., Cameron, S., Sass, P., Zoller, M. & Wigler, M. (1987)
Cell
50, 277–287]. Although they are redundant for viability, the three A kinases are not redundant for pseudohyphal growth [Robertson, L. S. & Fink, G. R. (1998)
Proc. Natl. Acad. Sci. USA
95, 13783–13787; Pan, X. & Heitman, J. (1999)
Mol. Cell. Biol.
19, 4874–4887]; Tpk2, but not Tpk1 or Tpk3, is required for pseudohyphal growth. Genome-wide transcriptional profiling has revealed unique signatures for each of the three A kinases leading to the identification of additional functional diversity among these proteins. Tpk2 negatively regulates genes involved in iron uptake and positively regulates genes involved in trehalose degradation and water homeostasis. Tpk1 is required for the derepression of branched chain amino acid biosynthesis genes that seem to have a second role in the maintenance of iron levels and DNA stability within mitochondria. The fact that
TPK2
mutants grow better than wild types on nonfermentable carbon sources and on media deficient in iron supports the unique role of Tpk2 in respiratory growth and carbon source use.
Bone patterning is altered in the regenerating zebrafish caudal fin after ectopic expression of
sonic hedgehog
and
bmp2b
or exposure to cyclopamine
Authors: Quint Elizabeth , Smith Amanda , Avaron Fabien , Laforest Lynda , Miles Jennifer , Gaffield William , Akimenko Marie-Andrée
Published: July 2002

Amputation of the zebrafish caudal fin stimulates regeneration of the dermal skeleton and reexpression of sonic hedgehog (shh)-signaling pathway genes. Expression patterns suggest a role for shh signaling in the secretion and patterning of the regenerating dermal bone, but a direct role has not been demonstrated. We established an
in vivo
method of gene transfection to express ectopically genes in the blastema of regenerating fins. Ectopic expression of
shh
or
bmp2
in the blastema-induced excess bone deposition and altered patterning of the regenerate. The effects of shh ectopic expression could be antagonized by ectopic expression of chordin, an inhibitor of bone morphogenetic protein (bmp) signaling. We disrupted shh signaling in the regenerating fin by exposure to cyclopamine and found a dose-dependent inhibition of fin outgrowth, accumulation of melanocytes in the distal region of each fin ray, loss of actinotrichia, and reduction in cell proliferation in the mesenchyme. Morphological changes were accompanied by an expansion, followed by a reduction, in domains of
shh
expression and a rapid abolition of
ptc1
expression. These results implicate shh and bmp2b signaling in the proliferation and/or differentiation of specialized bone-secreting cells in the blastema and suggest
shh
expression may be controlled by regulatory feedback mechanisms that define the region of bone secretion in the outgrowing fin.
NTP-sensing by rRNA promoters in
Escherichia coli
is direct
Authors: Schneider David A. , Gaal Tamas , Gourse Richard L.
Published: July 2002

We showed previously that
rrn
P1 promoters require unusually high concentrations of the initiating nucleoside triphosphates (ATP or GTP, depending on the promoter) for maximal transcription
in vitro
. We proposed that this requirement for high initiating NTP concentrations contributes to control of the
rrn
P1 promoters from the seven
Escherichia coli
rRNA operons. However, the previous studies did not prove that variation in NTP concentration affects
rrn
P1 promoter activity directly
in vivo
. Here, we create conditions
in vivo
in which ATP and GTP concentrations are altered in opposite directions relative to one another, and we show that transcription from
rrn
P1 promoters that initiate with either ATP or GTP follows the concentration of the initiating NTP for that promoter. These results demonstrate that the effect of initiating NTP concentration on
rrn
P1 promoter activity
in vivo
is direct. As predicted by a model in which homeostatic control of rRNA transcription results, at least in part, from sensing of NTP concentrations by
rrn
P1 promoters, we show that inhibition of protein synthesis results in an increase in ATP concentration and a corresponding increase in transcription from
rrnB
P1. We conclude that translation is a major consumer of purine NTPs, and that NTP-sensing by
rrn
P1 promoters serves as a direct regulatory link between translation and ribosome synthesis.
Intramolecular secondary structure rearrangement by the kissing interaction of the
Neurospora
VS ribozyme
Authors: Andersen Angela A. , Collins Richard A.
Published: July 2002

Kissing interactions in RNA are formed when bases between two hairpin loops pair. Intra- and intermolecular kissing interactions are important in forming the tertiary or quaternary structure of many RNAs. Self-cleavage of the wild-type Varkud satellite (VS) ribozyme requires a kissing interaction between the hairpin loops of stem-loops I and V. In addition, self-cleavage requires a rearrangement of several base pairs at the base of stem I. We show that the kissing interaction is necessary for the secondary structure rearrangement of wild-type stem-loop I. Surprisingly, isolated stem-loop V in the absence of the rest of the ribozyme is sufficient to rearrange the secondary structure of isolated stem-loop I. In contrast to kissing interactions in other RNAs that are either confined to the loops or culminate in an extended intermolecular duplex, the VS kissing interaction causes changes in intramolecular base pairs within the target stem-loop.
NMR investigation of ferricytochrome
c
unfolding: Detection of an equilibrium unfolding intermediate and residual structure in the denatured state
Authors: Russell Brandy S. , Melenkivitz Rory , Bren Kara L.
Published: July 2002

Horse ferricytochrome
c
(cyt
c
) undergoes exchange of one of its axial heme ligands (Met-80) for one or more non-native ligands under denaturing conditions. We have used
1
H NMR spectroscopy to detect two conformations of paramagnetic cyt
c
with non-native heme ligation through a range of urea concentrations. One non-native form is an equilibrium unfolding intermediate observed under partially denaturing conditions and is attributed to replacement of Met-80 with one or more Lys side chains. The second non-native form, in which the native Met ligand is replaced by a His, is observed under strongly denaturing conditions. Thermodynamic analysis of these data indicates a relatively small Δ
G
(17 kJ/mol) for the transition from native to the Lys-ligated intermediate and a significantly larger Δ
G
(47 kJ/mol) for the transition from native to the His-ligated species. Although CD and fluorescence data indicate that the equilibrium unfolding of cyt
c
is a two-state process, these NMR results implicate an intermediate with His-Lys ligation.
Purified hematopoietic stem cell grafts induce tolerance to alloantigens and can mediate positive and negative T cell selection
Authors: Shizuru Judith A. , Weissman Irving L. , Kernoff Rachel , Masek Marilyn , Scheffold Yolanda C.
Published: July 2002

Engraftment of allogeneic bone marrow (BM) has been shown to induce tolerance to organs genotypically matched with the BM donor. Immune reconstitution after BM transplantation therefore involves re-establishment of a T cell pool tolerant to antigens present on both donor and host tissues. However, how hematopoietic grafts exert their influence over the regenerating immune system is not completely understood. Prior studies suggest that education of the newly arising T cell pool involves distinct contributions from donor and host stromal elements. Specifically, negative selection is thought to be mediated primarily by donor BM-derived antigen-presenting cells, whereas positive selection is dictated by radio-resistant host-derived thymic stromal cells. In this report we studied the effect of highly purified allogeneic hematopoietic stem cells (HSCs) on organ transplantation tolerance induction and immune reconstitution. In contrast to engraftment of BM that results in near-complete donor T cell chimerism, HSC engraftment results in mixed T cell chimerism. Nonetheless we observed that HSC grafts induce tolerance to donor-matched neonatal heart grafts, and one way the HSC grafts alter host immune responses is via deletion of newly arising donor as well as radiation-resistant host T cells. Furthermore, using anin vivoassay of graft rejection to study positive selection we made the unexpected observation that T cells in chimeric mice rejected grafts only in the context of the donor MHC type. These latter findings conflict with the conventionally held view that radio-resistant host elements primarily dictate positive selection.
Human Trp3 forms both inositol trisphosphate receptor-dependent and receptor-independent store-operated cation channels in DT40 avian B lymphocytes
Authors: Vazquez Guillermo , Lievremont Jean-Philippe , St. J. Bird Gary , Putney James W.
Published: July 2002

Mammalian Trp proteins are candidates for plasma membrane calcium
channels regulated by receptor activation or by intracellular calcium
store depletion [capacitative calcium entry (CCE)]. One extensively
investigated member of the Trp family, the human Trp3 (hTrp3), behaves
as a receptor-activated, calcium-permeable, nonselective cation channel
when expressed in cell lines and does not appear to be activated by
store depletion. Nonetheless, there is good evidence that Trp3 can be
regulated by interacting with inositol trisphosphate receptors
(IP
3
Rs), reminiscent of the conformational coupling mode of
CCE. To investigate the role of Trp3 in CCE, and its regulation by
IP
3
R, we transiently expressed hTrp3 in the wild-type DT40
chicken B lymphocyte cell line and its variant lacking
IP
3
R. Expression of hTrp3 in either wild-type or
IP
3
R-knockout cells did not increase basal membrane
permeability, but resulted in a substantially greater divalent cation
entry after thapsigargin-induced store depletion. This hTrp3-dependent
divalent cation entry was significantly greater in the wild type than
in IP
3
R-knockout cells. Thus, it appears that in this cell
line, hTrp3 forms channels that are store-operated by both
IP
3
R-dependent and IP
3
R-independent mechanisms.
Trp3, or one of its structural relatives, is a candidate for the
store-operated, nonselective cation channels observed in smooth muscle
cells and other cell types.
Phosphorylation of murine p53 at Ser-18 regulates the p53 responses to DNA damage
Authors: Chao Connie , Saito Shin'ichi , Anderson Carl W. , Appella Ettore , Xu Yang
Published: July 2002

Ser-15 of human p53 (corresponding to Ser-18 of mouse p53) is
phosphorylated by ataxia-telangiectasia mutated (ATM) family kinases in
response to ionizing radiation (IR) and UV light. To determine the
effects of phosphorylation of endogenous murine p53 at
Ser-18 on biological responses to DNA damage, we introduced a missense
mutation (Ser-18 to Ala) by homologous recombination into both alleles
of the endogenous p53 gene in mouse embryonic stem (ES)
cells. Our analyses showed that phosphorylation of murine p53 at Ser-18
in response to IR or UV radiation was required for a full p53-mediated
response to these DNA damage-inducing agents. In contrast,
phosphorylation of p53 at Ser-18 was not required for ATM-dependent
cellular resistance after exposure to IR. Additionally, efficient
acetylation of the C terminus of p53 in response to DNA damage did not
require phosphorylation of murine p53 at Ser-18.
An essential amino acid induces epithelial β-defensin expression
Authors: Fehlbaum Pascale , Rao Meena , Zasloff Michael , Anderson G. Mark
Published: July 2002

Antimicrobial peptides constitute an important component of the mammalian innate immune response. Several types of antimicrobial peptides, including the β-defensins, are produced at epithelial surfaces in response to infectious threats. Here we show that a class of small molecules, includingl-isoleucine and several of its analogs, can specifically induce epithelial β-defensin expression. This induction is transcriptional in nature and involves activation of the NF-κB/rel family of trans-activating factors. We hypothesize that these substances represent unique markers for the presence of pathogens and are recognized by innate immune pattern recognition receptors. Isoleucine or its analogs ultimately may have clinical utility as novel immunostimulants that could bolster the barrier defenses of mucosal surfaces.
Tumor induction by an Lck-MyrAkt transgene is delayed by mechanisms controlling the size of the thymus
Authors: Malstrom Scott , Tili Esmerina , Kappes Dietmar , Ceci Jeffrey D. , Tsichlis Philip N.
Published: July 2002

Transgenic mice expressing MyrAkt from a proximal Lck promoter
construct develop thymomas at an early age, whereas transgenic
mice expressing constitutively active Lck-AktE40K develop
primarily tumors of the peripheral lymphoid organs later in life. The
thymus of 6- to 8-week-old MyrAkt transgenic mice is normal in size but
contains fewer, larger cells than the thymus of nontransgenic control
and AktE40K transgenic mice. Earlier studies had shown that cell size
and cell cycle are coordinately regulated. On the basis of this
finding, and our observations that the oncogenic potential of
Akt correlates with its effect on cell size, we hypothesized that
mechanisms aimed at maintaining the size of the thymus dissociate cell
size and cell cycle regulation by blocking MyrAkt-promoted
G
1
progression and that failure of these mechanisms may
promote cell proliferation resulting in an enlarged neoplastic thymus.
To address this hypothesis, we examined the cell cycle distribution of
freshly isolated and cultured thymocytes from transgenic and
nontransgenic control mice. The results showed that although neither
transgene alters cell cycle distribution
in situ
, the
MyrAkt transgene promotes G
1
progression in culture.
Freshly isolated MyrAkt thymocytes express high levels of cyclins D2
and E and cdk4 but lower than normal levels of cyclin D3 and cdk2.
Cultured thymocytes from MyrAkt transgenic mice, on the other hand,
express high levels of cyclin D3, suggesting that the hypothesized
organ size control mechanisms may down-regulate the expression of this
molecule. Primary tumor cells, similar to MyrAkt thymocytes in culture,
express high levels of cyclin D3. These findings support the hypothesis
that tumor induction is caused by the failure of organ size
control mechanisms to down-regulate cyclin D3 and to block
MyrAkt-promoted G
1
progression.
Balanced branching in transcription termination
Authors: Harrington K. J. , Laughlin R. B. , Liang S.
Published: July 2002

The theory of stochastic transcription termination based on
free-energy competition [von Hippel, P. H. & Yager, T. D.
(1992)
Science
255, 809–812 and von Hippel, P. H.
& Yager, T. D. (1991)
Proc. Natl. Acad. Sci.
USA
88, 2307–2311] requires two or more reaction rates
to be delicately balanced over a wide range of physical conditions. A
large body of work on glasses and large molecules suggests that this
balancing should be impossible in such a large system in the absence of
a new organizing principle of matter. We review the experimental
literature of termination and find no evidence for such a principle,
but do find many troubling inconsistencies, most notably, anomalous
memory effects. These effects suggest that termination has a
deterministic component and may conceivably not be stochastic at all.
We find that a key experiment by Wilson and von Hippel [Wilson,
K. S. & von Hippel, P. H. (1994)
J. Mol. Biol.
244, 36–51] thought to demonstrate stochastic termination was an
incorrectly analyzed regulatory effect of Mg
2+
binding.
Chlamydia trachomatis
cytotoxicity associated with complete and partial cytotoxin genes
Authors: Belland Robert J. , Scidmore Marci A. , Crane Deborah D. , Hogan Daniel M. , Whitmire William , McClarty Grant , Caldwell Harlan D.
Published: July 2002

Chlamydia trachomatis
is an obligate intracellular human bacterial pathogen that infects epithelial cells of the eye and genital tract. Infection can result in trachoma, the leading cause of preventable blindness worldwide, and sexually transmitted diseases. A common feature of infection is a chronic damaging inflammatory response for which the molecular pathogenesis is not understood. It has been proposed that chlamydiae have a cytotoxic activity that contributes to this pathology, but a toxin has not been identified. The
C. trachomatis
genome contains genes that encode proteins with significant homology to large clostridial cytotoxins. Here we show that
C. trachomatis
makes a replication-independent cytotoxic activity that produces morphological and cytoskeletal changes in epithelial cells that are indistinguishable from those mediated by clostridial toxin B. A mouse chlamydial strain that encodes a full-length cytotoxin caused pronounced cytotoxicity, as did a human strain that has a shorter ORF with homology to only the enzymatically active site of clostridial toxin B. Cytotoxin gene transcripts were detected in chlamydiae-infected cells, and a protein with the expected molecular mass was present in lysates of infected epithelial cells. The protein was present transiently in infected cells during the period of cytotoxicity. Together, these data provide compelling evidence for a chlamydial cytotoxin for epithelial cells and imply that the cytotoxin is present in the elementary body and delivered to host cells very early during infection. We hypothesize that the cytotoxin is a virulence factor that contributes to the pathogenesis of
C. trachomatis
diseases.
Candidate odorant receptors from the malaria vector mosquito
Anopheles gambiae
and evidence of down-regulation in response to blood feeding
Authors: Fox A. N. , Pitts R. J. , Robertson H. M. , Carlson J. R. , Zwiebel L. J.
Published: July 2002

Olfaction plays a major role in host preference and blood
feeding, integral behaviors for disease transmission by the malaria
vector mosquito
Anopheles gambiae
sensu stricto
(henceforth
A. gambiae).
We have identified four genes
encoding candidate odorant receptors from
A. gambiae
that are selectively expressed in olfactory organs, contain
approximately seven transmembrane domains, and show significant
similarity to several putative odorant receptors in
Drosophila
melanogaster
. Furthermore, one of the putative
A.
gambiae
odorant receptors exhibits female-specific antennal
expression and is down-regulated 12 h after blood feeding, a
period during which substantial reduction in olfactory responses to
human odorants has been observed. Taken together, these data suggest
these genes encode a family of odorant receptors in
A.
gambiae,
whose further study may aid in the design of novel
antimalarial programs.
Immunoregulatory properties of ISG15, an interferon-induced cytokine.
Authors: D'Cunha J , Knight E , Haas A L , Truitt R L , Borden E C
Published: July 2002

ISG15 is a 15-kDa protein of unique primary amino acid sequence, which is transcriptionally regulated by interferon (IFN) alpha and IFN-beta. Because it is synthesized in many cell types and secreted from human monocytes and lymphocytes, we postulated that ISG15 might act to modulate immune cell function. ISG15 stimulated B-depleted lymphocyte proliferation in a dose-dependent manner with significant proliferation induced by amounts of ISG15 as low as 1 ng/ml (58 pM). Maximal stimulation of [3H]thymidine incorporation by B-depleted lymphocytes occurred at 6-7 days. Immunophenotyping of ISG15-treated B-depleted lymphocyte cultures indicated a 26-fold expansion of natural killer (NK) cells (CD56+). In cytotoxicity assays, ISG15 was a potent inducer of cytolytic activity directed against both K562 (100 lytic units per 10(6) cells) and Daudi (80 lytic units per 10(6) cells) tumor cell targets, indicating that ISG15 enhanced lymphokine-activated killer-like activity. ISG15-induced NK cell proliferation required coculturing of T and NK cells, suggesting that soluble factor(s) were required. Measurement of ISG15-treated cell culture supernatants for cytokines indicated production of IFN-gamma (> 700 units/ml). No interleukin 2 or interleukin 12 was detected. IFN-gamma itself failed to stimulate lymphocyte proliferation and lymphokine-activated killer cell activation. Further, induced expression of IFN-gamma mRNA was detected by reverse transcription-PCR in T lymphocytes after ISG15 treatment but not in NK cells. Enhancement of NK cell proliferation, augmentation of non-major histocompatibility complex-restricted cytotoxicity, and induction of IFN-gamma from T cells identify ISG15 as a member of the cytokine cascade and suggest that it may be responsible for amplifying and directing some of the immunomodulatory effects of IFN-alpha or IFN-beta.
Msn2p, a zinc finger DNA-binding protein, is the transcriptional activator of the multistress response in Saccharomyces cerevisiae.
Authors: Schmitt A P , McEntee K
Published: July 2002

The stress response promoter element (STRE) confers increased transcription to a set of genes following environmental or metabolic stress in Saccharomyces cerevisiae. A lambda gt11 library was screened to isolate clones encoding STRE-binding proteins, and one such gene was identified as MSN2, which encoded a zinc-finger transcriptional activator. Disruption of the MSN2 gene abolished an STRE-binding activity in crude extracts as judged by both gel mobility-shift and Southwestern blot experiments, and overexpression of MSN2 intensified this binding activity. Northern blot analysis demonstrated that for the known or suspected STRE-regulated genes DDR2, CTT1, HSP12, and TPS2, transcript induction was impaired following heat shock or DNA damage treatment in the msn2-disrupted strain and was constitutively activated in a strain overexpressing MSN2. Furthermore, heat shock induction of a STRE-driven reporter gene was reduced more than 6-fold in the msn2 strain relative to wild-type cells. Taken together, these data indicate that Msn2p is the transcription factor that activates STRE-regulated genes in response to stress. Whereas nearly 85% of STRE-mediated heat shock induction was MSN2 dependent, there was significant MSN2-independent expression. We present evidence that the MSN2 homolog, MSN4, can partially replace MSN2 for transcriptional activation following stress. Moreover, our data provides evidence for the involvement of additional transcription factors in the yeast multistress response.
A mechanism for inducing plant development: the genesis of a specific inhibitor.
Authors: Smith C E , Ruttledge T , Zeng Z , O'Malley R C , Lynn D G
Published: July 2002

Parasitic strategies are widely distributed in the plant kingdom and frequently involve coupling parasite organogenesis with cues from the host. In Striga asiatica, for example, the cues that initiate the development of the host attachment organ, the haustorium, originate in the host and trigger the transition from vegetative to parasitic mode in the root meristem. This system therefore offers a unique opportunity to study the signals and mechanisms that control plant cell morphogenesis. Here we establish that the biological activity of structural analogs of the natural inducer displays a marked dependence on redox potential and suggest the existence of a semiquinone intermediate. Building on chemistry that exploits the energetics of such an intermediate, cyclopropyl-p-benzoquinone (CPBQ) is shown to be a specific inhibitor of haustorial development. These data are consistent with a model where haustorial development is initiated by the completion of a redox circuit.
Deficits in memory and hippocampal long-term potentiation in mice with reduced calbindin D28K expression.
Authors: Molinari S , Battini R , Ferrari S , Pozzi L , Killcross A S , Robbins T W , Jouvenceau A , Billard J M , Dutar P , Lamour Y , Baker W A , Cox H , Emson P C
Published: July 2002

The influx of calcium into the postsynaptic neuron is likely to be an important event in memory formation. Among the mechanisms that nerve cells may use to alter the time course or size of a spike of intracellular calcium are cytosolic calcium binding or "buffering" proteins. To consider the role in memory formation of one of these proteins, calbindin D28K, which is abundant in many neurons, including the CA1 pyramidal cells of the hippocampus, transgenic mice deficient in calbindin D28K have been created. These mice show selective impairments in spatial learning paradigms and fail to maintain long-term potentiation. These results suggest a role for calbindin D28K protein in temporally extending a neuronal calcium signal, allowing the activation of calcium-dependent intracellular signaling pathways underlying memory function.
Z-DNA-forming sites within the human beta-globin gene cluster.
Authors: Müller V , Takeya M , Brendel S , Wittig B , Rich A
Published: July 2002

Agarose-encapsulated, metabolically active, permeabilized nuclei from human hematopoietic cell lines were tested for Z-DNA formation in the beta-globin gene cluster. Biotinylated monoclonal antibodies against Z-DNA were diffused into the nuclei and cross-linked to DNA with a 10-ns laser exposure at 266 nm. Following digestion with restriction enzymes, fragments that had formed Z-DNA were isolated. Seventeen regions with Z-DNA sequence motifs in the 73-kb region were studied by PCR amplification, and five were found in the Z conformation.
Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins.
Authors: Mesnil M , Piccoli C , Tiraby G , Willecke K , Yamasaki H
Published: July 2002

In gene therapy to treat cancer, typically only a fraction of the tumor cells can be successfully transfected with a gene. However, in the case of brain tumor therapy with the thymidine kinase gene from herpes simplex virus (HSV-tk), not only the cells transfected with the gene but also neighboring others can be killed in the presence of ganciclovir. Such a "bystander" effect is reminiscent of our previous observation that the effect of certain therapeutic agents may be enhanced by their diffusion through gap junctional intercellular communication (GJIC). Herein, we present the evidence, from in vitro studies, that gap junctions could indeed be responsible for such a gene therapy bystander effect. We used HeLa cells for this purpose, since they show very little, if any, ability to communicate through gap junctions. When HeLa cells were transfected with HSV-tk gene and cocultured with nontransfected cells, only HSV-tk-transfected HeLa cells (tk+) were killed by ganciclovir. However, when HeLa cells transfected with a gene encoding for the gap junction protein, connexin 43 (Cx43), were used, not only tk+ cells, but also tk- cells were killed, presumably due to the transfer, via Cx43-mediated GJIC, of toxic ganciclovir molecules phosphorylated by HSV-tk to the tk- cells. Such bystander effect was not observed when tk+ and tk- cells were cocultured without direct cell-cell contact between those two types of cells. Thus, our results give strong evidence that the bystander effect seen in HSV-tk gene therapy may be due to Cx-mediated GJIC.