bluuebunny/crossref_metadata_2025 huggingface dataset:
Discrete and polymeric self-assembled dendrimers: Hydrogen bond-mediated assembly with high stability and high fidelity
Authors: Corbin Perry S. , Lawless Laurence J. , Li Zhanting , Ma Yuguo , Witmer Melissa J. , Zimmerman Steven C.
Published: July 2002

Hydrogen bond-mediated self-assembly is a powerful strategy for creating nanoscale structures. However, little is known about the fidelity of assembly processes that must occur when similar and potentially competing hydrogen-bonding motifs are present. Furthermore, there is a continuing need for new modules and strategies that can amplify the relatively weak strength of a hydrogen bond to give more stable assemblies. Herein we report quantitative complexation studies on a ureidodeazapterin-based module revealing an unprecedented stability for dimers of its self-complementary acceptoracceptor-donor-donor (AADD) array. Linking two such units together with a semirigid spacer that carries a first-, second-, or third-generation Fréchet-type dendron affords a ditopic structure programmed to self assemble. The specific structure that is formed depends both on the size of the dendron and the solvent, but all of the assemblies have exceptionally high stability. The largest discrete nanoscale assembly is a hexamer with a molecular mass of about 17.8 kDa. It is stabilized by 30 hydrogen bonds, including six AADD⋅DDAA contacts. The hexamer forms and is indefinitely stable in the presence of a hexamer containing six ADD⋅DAA hydrogen-bonding arrays.
Gregarious behavior in desert locusts is evoked by touching their back legs
Authors: Simpson S. J. , Despland E. , Hägele B. F. , Dodgson T.
Published: July 2002

Desert locusts in the solitarious phase were repeatedly touched on
various body regions to identify the site of mechanosensory input that
elicits the transition to gregarious phase behavior. The phase state of
individual insects was measured after a 4-h period of localized
mechanical stimulation, by using a behavioral assay based on multiple
logistic regression analysis. A significant switch from solitarious to
gregarious behavior occurred when the outer face of a hind femur had
been stimulated, but mechanical stimulation of 10 other body regions
did not result in significant behavioral change. We conclude that a
primary cause of the switch in behavior that seeds the formation of
locust swarms is individuals regularly touching others on the hind legs
within populations that have become concentrated by the environment.
Global survey of genetic variation in
CCR5
,
RANTES
, and
MIP-1
α: Impact on the epidemiology of the HIV-1 pandemic
Authors: Gonzalez Enrique , Dhanda Rahul , Bamshad Mike , Mummidi Srinivas , Geevarghese Reni , Catano Gabriel , Anderson Stephanie A. , Walter Elizabeth A. , Stephan Kevin T. , Hammer Michael F. , Mangano Andrea , Sen Luisa , Clark Robert A. , Ahuja Seema S. , Dolan Matthew J. , Ahuja Sunil K.
Published: July 2002

Expression of CC chemokine receptor 5 (CCR5), the major
coreceptor for HIV-1 cell entry, and its ligands (e.g., RANTES and
MIP-1α) is widely regarded as central to the pathogenesis of HIV-1
infection. By surveying nearly 3,000 HIV+ and HIV− individuals from
worldwide populations for polymorphisms in the genes encoding RANTES,
MIP-1α, and CCR5, we show that the evolutionary histories of human
populations have had a significant impact on the distribution of
variation in these genes, and that this may be responsible, in part,
for the heterogeneous nature of the epidemiology of the HIV-1 pandemic.
The varied distribution of
RANTES
haplotypes (AC, GC,
and AG) associated with population-specific HIV-1 transmission- and
disease-modifying effects is a striking example. Homozygosity for the
AC haplotype was associated with an increased risk of acquiring HIV-1
as well as accelerated disease progression in European Americans, but
not in African Americans. Yet, the prevalence of the ancestral AC
haplotype is high in individuals of African origin, but substantially
lower in non-Africans. In a Japanese cohort, AG-containing
RANTES
haplotype pairs were associated with a delay in
disease progression; however, we now show that their contribution to
HIV-1 pathogenesis and epidemiology in other parts of the world is
negligible because the AG haplotype is infrequent in non-Far East
Asians. Thus, the varied distribution of
RANTES
,
MIP-1
α, and
CCR5
haplotype pairs and
their population-specific phenotypic effects on HIV-1 susceptibility
and disease progression results in a complex pattern of biological
determinants of HIV-1 epidemiology. These findings have important
implications for the design, assessment, and implementation
of effective HIV-1 intervention and prevention strategies.
Organic semiconductors: A theoretical characterization of the basic parameters governing charge transport
Authors: Brédas J. L. , Calbert J. P. , da Silva Filho D. A. , Cornil J.
Published: July 2002

Organic semiconductors based on π-conjugated oligomers and polymers constitute the active elements in new generations of plastic (opto)electronic devices. The performance of these devices depends largely on the efficiency of the charge-transport processes; at the microscopic level, one of the major parameters governing the transport properties is the amplitude of the electronic transfer integrals between adjacent oligomer or polymer chains. Here, quantum-chemical calculations are performed on model systems to address the way transfer integrals between adjacent chains are affected by the nature and relative positions of the interacting units. Compounds under investigation include oligothienylenes, hexabenzocoronene, oligoacenes, and perylene. It is shown that the amplitude of the transfer integrals is extremely sensitive to the molecular packing. Interestingly, in contrast to conventional wisdom, specific arrangements can lead to electron mobilities that are larger than hole mobilities, which is, for instance, the case of perylene.
Granzymes A and B directly cleave lamins and disrupt the nuclear lamina during granule-mediated cytolysis
Authors: Zhang Dong , Beresford Paul J. , Greenberg Arnold H. , Lieberman Judy
Published: July 2002

Cytotoxic T lymphocytes (CTL) induce apoptosis by engaging death receptors or by exocytosis of cytolytic granules containing granzyme (Gzm) proteases and perforin. The lamins, which maintain the structural integrity of the nuclear envelope, are cleaved by caspases during caspase-mediated apoptosis. Although death receptor engagement and GzmB activate caspases, CTL also induce apoptosis during caspase blockade. Both GzmA and GzmB directly and efficiently cleave laminBin vitro,in situin isolated nuclei and in cells loaded with perforin and Gzms, even in the presence of caspase inhibitors. LaminB is cleaved by GzmA at concentrations of 3 nM, but GzmB is 50 times less active. GzmA cuts laminB at R392; GzmB cuts at the caspase VEVD231 site. Characteristic laminB fragments generated by Gzm proteolysis also are observed during CTL lysis, even in the presence of caspase inhibitors or in cells overexpressing bcl-2. Lamins A/C are direct substrates of GzmA, but not GzmB. GzmA and GzmB therefore directly target critical caspase substrates in caspase-resistant cells.
Interaction of the β sliding clamp with MutS, ligase, and DNA polymerase I
Authors: López de Saro Francisco J. , O'Donnell Mike
Published: July 2002

The β and proliferating cell nuclear antigen (PCNA) sliding clamps were first identified as components of their respective replicases, and thus were assigned a role in chromosome replication. Further studies have shown that the eukaryotic clamp, PCNA, interacts with several other proteins that are involved in excision repair, mismatch repair, cellular regulation, and DNA processing, indicating a much wider role than replication alone. Indeed, the
Escherichia coli
β clamp is known to function with DNA polymerases II and V, indicating that β also interacts with more than just the chromosomal replicase, DNA polymerase III. This report demonstrates three previously undetected protein–protein interactions with the β clamp. Thus, β interacts with MutS, DNA ligase, and DNA polymerase I. Given the diverse use of these proteins in repair and other DNA transactions, this expanded list of β interactive proteins suggests that the prokaryotic β ring participates in a wide variety of reactions beyond its role in chromosomal replication.
Prediction of cognitive decline in normal elderly subjects with 2-[
18
F]fluoro-2-deoxy-
d
-glucose/positron-emission tomography (FDG/PET)
Authors: de Leon M. J. , Convit A. , Wolf O. T. , Tarshish C. Y. , DeSanti S. , Rusinek H. , Tsui W. , Kandil E. , Scherer A. J. , Roche A. , Imossi A. , Thorn E. , Bobinski M. , Caraos C. , Lesbre P. , Schlyer D. , Poirier J. , Reisberg B. , Fowler J.
Published: July 2002

Neuropathology studies show that patients with mild cognitive impairment (MCI) and Alzheimer's disease typically have lesions of the entorhinal cortex (EC), hippocampus (Hip), and temporal neocortex. Related observations with
in vivo
imaging have enabled the prediction of dementia from MCI. Although individuals with normal cognition may have focal EC lesions, this anatomy has not been studied as a predictor of cognitive decline and brain change. The objective of this MRI-guided 2-[
18
F]fluoro-2-deoxy-
d
-glucose/positron-emission tomography (FDG/PET) study was to examine the hypothesis that among normal elderly subjects, EC METglu reductions predict decline and the involvement of the Hip and neocortex. In a 3-year longitudinal study of 48 healthy normal elderly, 12 individuals (mean age 72) demonstrated cognitive decline (11 to MCI and 1 to Alzheimer's disease). Nondeclining controls were matched on apolipoprotein E genotype, age, education, and gender. At baseline, metabolic reductions in the EC accurately predicted the conversion from normal to MCI. Among those who declined, the baseline EC predicted longitudinal memory and temporal neocortex metabolic reductions. At follow-up, those who declined showed memory impairment and hypometabolism in temporal lobe neocortex and Hip. Among those subjects who declined, apolipoprotein E E4 carriers showed marked longitudinal temporal neocortex reductions. In summary, these data suggest that an EC stage of brain involvement can be detected in normal elderly that predicts future cognitive and brain metabolism reductions. Progressive E4-related hypometabolism may underlie the known increased susceptibility for dementia. Further study is required to estimate individual risks and to determine the physiologic basis for METglu changes detected while cognition is normal.
Localized cerebral blood flow response at submillimeter columnar resolution
Authors: Duong Timothy Q. , Kim Dae-Shik , Uğurbil Kâmil , Kim Seong-Gi
Published: July 2002

Functional magnetic resonance imaging (fMRI) has been widely used for imaging brain functions. However, the extent of the fMRI hemodynamic response around the active sites, at submillimeter resolution, remains poorly understood and controversial. With the use of perfusion-based fMRI, we evaluated the hemodynamic response in the cat visual cortex after orientation-specific stimuli. Activation maps obtained by using cerebral blood flow fMRI measurements were predominantly devoid of large draining vein contamination and reproducible at columnar resolution. Stimulus-specific cerebral blood flow responses were spatially localized to individual cortical columns, and columnar layouts were resolved. The periodic spacing of orientation columnar structures was estimated to be 1.1 ± 0.2 mm (
n
= 14 orientations, five animals), consistent with previous findings. The estimated cerebral blood flow response at full width at half-maximum was 470 μm under single-stimulus conditions without differential subtraction. These results suggest that hemodynamic-based fMRI can indeed be used to map individual functional columns if large-vessel contributions can be minimized or eliminated.
A possible loophole in the theorem of Bell
Authors: Hess Karl , Philipp Walter
Published: July 2002

The celebrated inequalities of Bell are based on the
assumption that local hidden parameters exist. When combined with
conflicting experimental results, these inequalities appear to prove
that local hidden parameters cannot exist. This contradiction
suggests to many that only instantaneous action at a distance can
explain the Einstein, Podolsky, and Rosen type of experiments. We show
that, in addition to the assumption that hidden parameters exist, Bell
tacitly makes a variety of other assumptions that contribute to his
being able to obtain the desired contradiction. For instance, Bell
assumes that the hidden parameters do not depend on time and are
governed by a single probability measure independent of the analyzer
settings. We argue that the exclusion of time has neither a physical
nor a mathematical basis but is based on Bell's translation of the
concept of Einstein locality into the language of probability theory.
Our additional set of local hidden variables includes time-like
correlated parameters and a generalized probability density. We prove
that our extended space of local hidden variables does not permit
Bell-type proofs to go forward.
Behavioral genetics of thermosensation and hygrosensation in Drosophila.
Authors: Sayeed O , Benzer S
Published: July 2002

Whereas temperature and humidity are critical variables affecting physiology, behavior, and evolution, the genetic and neuronal underpinnings of thermosensation and hygrosensation remain poorly understood. We have initiated a behavioral-genetic investigation of these sensory systems in Drosophila. Behavioral tests are described for the rapid screening of mutants defective in thermosensation and hygrosensation. We demonstrate the strong responses of normal flies to temperature and humidity. Two mutants were found with defects in thermosensation, only one of which is also defective in hygrosensation, indicating that they involve different sensory mechanisms. Ablation experiments further separate these sensory systems by showing that thermoreceptors are housed in the third antennal segment, whereas hygroreceptors are located more distally in the antennal arista.
Engineering actin-resistant human DNase I for treatment of cystic fibrosis.
Authors: Ulmer J S , Herzka A , Toy K J , Baker D L , Dodge A H , Sinicropi D , Shak S , Lazarus R A
Published: July 2002

Human deoxyribonuclease I (DNase I), an enzyme recently approved for treatment of cystic fibrosis (CF), has been engineered to create two classes of mutants: actin-resistant variants, which still catalyze DNA hydrolysis but are no longer inhibited by globular actin (G-actin) and active site variants, which no longer catalyze DNA hydrolysis but still bind G-actin. Actin-resistant variants with the least affinity for actin, as measured by an actin binding ELISA and actin inhibition of [33P] DNA hydrolysis, resulted from the introduction of charged, aliphatic, or aromatic residues at Ala-114 or charged residues on the central hydrophobic actin binding interface at Tyr-65 or Val-67. In CF sputum, the actin-resistant variants D53R, Y65A, Y65R, or V67K were 10-to 50-fold more potent than wild type in reducing viscoelasticity as determined in sputum compaction assays. The reduced viscoelasticity correlated with reduced DNA length as measured by pulsed-field gel electrophoresis. In contrast, the active site variants H252A or H134A had no effect on altering either viscoelasticity or DNA length in CF sputum. The data from both the active site and actin-resistant variants demonstrate that the reduction of viscoelasticity by DNase I results from DNA hydrolysis and not from depolymerization of filamentous actin (F-actin). The increased potency of the actin-resistant variants indicates that G-actin is a significant inhibitor of DNase I in CF sputum. These results further suggest that actin-resistant DNase I variants may have improved efficacy in CF patients.
Evidence for an insulin receptor substrate 1 independent insulin signaling pathway that mediates insulin-responsive glucose transporter (GLUT4) translocation.
Authors: Morris A J , Martin S S , Haruta T , Nelson J G , Vollenweider P , Gustafson T A , Mueckler M , Rose D W , Olefsky J M
Published: July 2002

Interaction of the activated insulin receptor (IR) with its substrate, insulin receptor substrate 1 (IRS-1), via the phosphotyrosine binding domain of IRS-1 and the NPXY motif centered at phosphotyrosine 960 of the IR, is important for IRS-1 phosphorylation. We investigated the role of this interaction in the insulin signaling pathway that stimulates glucose transport. Utilizing microinjection of competitive inhibitory reagents in 3T3-L1 adipocytes, we have found that disruption of the IR/IRS-1 interaction has no effect upon translocation of the insulin-responsive glucose transporter (GLUT4). The activity of these reagents was demonstrated by their ability to block insulin stimulation of two distinct insulin bioeffects, membrane ruffling and mitogenesis, in 3T3-L1 adipocytes and insulin-responsive rat 1 fibroblasts. These data suggest that phosphorylated IRS-1 is not an essential component of the metabolic insulin signaling pathway that leads to GLUT4 translocation, yet it appears to be required for other insulin bioeffects.
Resonant tectorial membrane motion in the inner ear: its crucial role in frequency tuning.
Authors: Gummer A W , Hemmert W , Zenner H P
Published: July 2002

The tectorial membrane has long been postulated as playing a role in the exquisite sensitivity of the cochlea. In particular, it has been proposed that the tectorial membrane provides a second resonant system, in addition to that of the basilar membrane, which contributes to the amplification of the motion of the cochlear partition. Until now, technical difficulties had prevented vibration measurements of the tectorial membrane and, therefore, precluded direct evidence of a mechanical resonance. In the study reported here, the vibration of the tectorial membrane was measured in two orthogonal directions by using a novel method of combining laser interferometry with a photodiode technique. It is shown experimentally that the motion of the tectorial membrane is resonant at a frequency of 0.5 octave (oct) below the resonant frequency of the basilar membrane and polarized parallel to the reticular lamina. It is concluded that the resonant motion of the tectorial membrane is due to a parallel resonance between the mass of the tectorial membrane and the compliance of the stereocilia of the outer hair cells. Moreover, in combination with the contractile force of outer hair cells, it is proposed that inertial motion of the tectorial membrane provides the necessary conditions to allow positive feedback of mechanical energy into the cochlear partition, thereby amplifying and tuning the cochlear response.
Local production of the p40 subunit of interleukin 12 suppresses T-helper 1-mediated immune responses and prevents allogeneic myoblast rejection.
Authors: Kato K , Shimozato O , Hoshi K , Wakimoto H , Hamada H , Yagita H , Okumura K
Published: July 2002

The p40 subunit of interleukin 12 (IL-12p40) has been known to act as an IL-12 antagonist in vitro. We here describe the immunosuppressive effect of IL-12p40 in vivo. A murine myoblast cell line, C2C12, was transduced with retro-virus vectors carrying the lacZ gene as a marker and the IL-12p40 gene. IL-12p40 secreted from the transfectant inhibited the IL-12-induced interferon gamma (IFN-gamma) production by splenocytes in vitro. Survival of C2C12 transplanted into allogeneic recipients was substantially prolonged when transduced with IL-12p40. Cytokine (IL-2 and IFN-gamma) production and cytotoxic T lymphocyte induction against allogeneic C2C12 were impaired in the recipients transplanted with the IL-12p40 transfectant. Delayed-type hypersensitivity response against C2C12 was also diminished in the IL-12p40 recipients. Furthermore, serum antibodies against beta-galactosidase of the T-helper 1-dependent isotypes (IgG2 and IgG3) were decreased in the IL-12p40 recipients. These results indicate that locally produced IL-12p40 exerts a potent immunosuppressive effect on T-helper 1-mediated immune responses that lead to allograft rejection. Therefore, IL-12p40 gene transduction would be useful for preventing the rejection of allografts and genetically modified own cells that are transduced with potentially antigenic molecules in gene therapy.
Suppression of tumorigenicity by the wild-type tuberous sclerosis 2 (Tsc2) gene and its C-terminal region.
Authors: Jin F , Wienecke R , Xiao G H , Maize J C , DeClue J E , Yeung R S
Published: July 2002

The Tsc2 gene, which is mutationally inactivated in the germ line of some families with tuberous sclerosis, encodes a large, membrane-associated GTPase activating protein (GAP) designated tuberin. Studies of the Eker rat model of hereditary cancer strongly support the role of Tsc2 as a tumor suppressor gene. In this study, the biological activity of tuberin was assessed by expressing the wild-type Tsc2 gene in tumor cell lines lacking functional tuberin and also in rat fibroblasts with normal levels of endogenous tuberin. The colony forming efficiency of Eker rat-derived renal carcinoma cells was significantly reduced following reintroduction of wild-type Tsc2. Tumor cells expressing the transfected Tsc2 gene became more anchorage-dependent and lost their ability to form tumors in severe combined immunodeficient mice. At the cellular level, restoration of tuberin expression caused morphological changes characterized by enlargement of the cells and increased contact inhibition. As with the full-length Tsc2 gene, a clone encoding only the C terminus of tuberin (amino acids 1049-1809, including the GAP domain) was capable of reducing both colony formation and in vivo tumorigenicity when transfected into the Eker rat tumor cells. In normal Rat1 fibroblasts, conditional overexpression of tuberin also suppressed colony formation and cell growth in vitro. These results provide direct experimental evidence for the tumor suppressor function of Tsc2 and suggest that the tuberin C terminus plays an important role in this activity.
Incremental lines of von Ebner in dinosaurs and the assessment of tooth replacement rates using growth line counts
Authors: Erickson G. M.
Published: July 2002

Dinosaur dentine exhibits growth lines that are tens of micrometers
in width. These laminations are homologous to incremental lines of von
Ebner found in extant mammal and crocodilian teeth (i.e., those of
amniotes). The lines likely reflect daily dentine formation, and they
were used to infer tooth development and replacement rates. In general,
dinosaur tooth formation rates negatively correlated with tooth size.
Theropod tooth replacement rates negatively correlated with tooth size,
which was due to limitations in the dentine formation rates of their
odontoblasts. Derived ceratopsian and hadrosaurian dinosaurs retained
relatively rapid tooth replacement rates through ontogeny. The
evolution of dental batteries in hadrosaurs and ceratopsians can be
explained by dentine formation constraints and rapid tooth wear. In
combination with counts of shed dinosaur teeth, tooth replacement rate
data can be used to assess population demographics of Mesozoic
ecosystems. Finally, it is of historic importance to note that Richard
Owen appears to have been the first to observe incremental lines of von
Ebner in dinosaurs more than 150 years ago.
beta2 knockout mice develop parenchymal iron overload: A putative role for class I genes of the major histocompatibility complex in iron metabolism.
Authors: Rothenberg B E , Voland J R
Published: July 2002

Hemochromatosis (HC) is an inherited disorder of iron absorption, mapping within the human major histocompatibility complex (MHC). We have identified a multigene system in the murine MHC that contains excellent candidates for the murine equivalent of the human HC locus and implicate nonclassical class I genes in the control of iron absorption. This gene system is characterized by multiple copies of two head-to-head genes encoded on opposite strands and driven by one common regulatory motif. This regulatory motif has a striking homology to the promoter region of the beta-globin gene, a gene obviously involved in iron metabolism and hence termed beta-globin analogous promoter (betaGAP). Upstream of the betaGAP sequence are nonclassical class I genes. At least one of these nonclassical class I genes, Q2, is expressed in the gastrointestinal tract, the primary site of iron absorption. Also expressed in the gastrointestinal tract and downstream of the betaGAP motif is a second set of putative genes, termed Hephaestus (HEPH). Based on these observations, we hypothesized that the genes that seem to be controlled by the betaGAP regulatory motifs would be responsible for the control of Fe absorption. As a test of this hypothesis, we predicted that mice which have altered expression of class I gene products, the beta2-microglobulin knockout mice, [beta2m(-/-)], would develop Fe overload. This prediction was confirmed, and these results indicate beta2m-associated proteins are involved in the control of intestinal Fe absorption.
A novel multifunctional
O
-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine
Authors: Li Laigeng , Popko Jacqueline L. , Zhang Xing-Hai , Osakabe Keishi , Tsai Chung-Jui , Joshi Chandrashekhar P. , Chiang Vincent L.
Published: July 2002

S
-adenosyl-
l
-methionine (SAM)-dependent
O
-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-
O
-methyltransferases (CAOMTs) and caffeoyl CoA 3-
O
-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (
Pinus taeda
) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic
A
cids/hydroxycinnamoyl CoA
E
sters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem.
Up-regulation of specific tyrosinase mRNAs in mouse melanomas with the
c

2j

gene substituted for the wild-type tyrosinase allele: Utilization in design of syngeneic immunotherapy models
Authors: Le Fur Nathalie , Silvers Willys K. , Kelsall Stephen R. , Mintz Beatrice
Published: July 2002

The expression of cell-specialization genes is likely to be changing in tumor cells as their differentiation declines. Functional changes in these genes might yield unusual peptide epitopes with anti-tumor potential and could occur without modification in the DNA sequence of the gene. Melanomas undergo a characteristic decline in melanization that may reflect altered contributions of key melanocytic genes such as tyrosinase. Quantitative reverse transcriptase–PCR of the wild-type (
C
) tyrosinase gene in transgenic (C57BL/6 strain) mouse melanomas has revealed a shift toward alternative splicing of the pre-mRNA that generated increased levels of the Δ1b and Δ1d mRNA splice variants. The spontaneous
c

2j

albino mutation of tyrosinase (in the C57BL/6 strain) changes the pre-mRNA splicing pattern. In
c

2j

/c

2j

melanomas, alternative splicing was again increased. However, while some mRNAs (notably Δ1b) present in
C/C
were obligatorily absent, others (Δ3 and Δ1d) were elevated. In
c

2j

/c

2j

melanomas, the percentage of total tyrosinase transcripts attributable to Δ3 reached approximately 2-fold the incidence in
c

2j

/c

2j

or
C/C
skin melanocytes. The percentage attributable to Δ1d rose to approximately 2-fold the incidence in
c

2j

/c

2j

skin, and to 10-fold that in
C/C
skin. These differences provide a basis for unique mouse models in which the melanoma arises in skin grafted from a
C/C
or
c

2j

/c

2j

transgenic donor to a transgenic host of the same or opposite tyrosinase genotype. Immunotherapy designs then could be based on augmenting those antigenic peptides that are novel or overrepresented in a tumor relative to the syngeneic host.
A role for TFIIH in controlling the activity of early RNA polymerase II elongation complexes
Authors: Dvir Arik , Conaway Ronald C. , Conaway Joan Weliky
Published: July 2002

TFIIH is a multifunctional RNA polymerase II transcription factor that possesses DNA-dependent ATPase, DNA helicase, and protein kinase activities. Previous studies have established that TFIIH enters the preinitiation complex and fulfills a critical role in initiation by catalyzing ATP-dependent formation of the open complex prior to synthesis of the first phosphodiester bond of nascent transcripts. In this report, we present direct evidence that TFIIH also controls RNA polymerase II activity at a postinitiation stage of transcription, by preventing premature arrest by very early elongation complexes just prior to their transition to stably elongating complexes. Unexpectedly, we observe that TFIIH is capable of entering the transcription cycle not only during assembly of the preinitiation complex but also after initiation and synthesis of as many as four to six phosphodiester bonds. These findings shed new light on the role of TFIIH in initiation and promoter escape and reveal an unanticipated flexibility in the ability of TFIIH to interact with RNA polymerase II transcription intermediates prior to, during, and immediately after initiation.
Reduction of HIV-1 in blood and lymph nodes following potent antiretroviral therapy and the virologic correlates of treatment failure
Authors: Wong Joseph K. , Günthard Huldrych F. , Havlir Diane V. , Zhang Zhi-Qiang , Haase Ashley T. , Ignacio Caroline C. , Kwok Shirley , Emini Emilio , Richman Douglas D.
Published: July 2002

Potent antiretroviral therapy can reduce plasma HIV RNA levels below the threshold of detection for periods of a year or more. The magnitude of HIV RNA reduction in the lymphoid tissue in patients with suppression of HIV RNA levels in plasma beyond 6 months has not been determined. We evaluated levels of HIV RNA and DNA and characterized resistance mutations in blood and inguinal lymph node biopsies obtained from 10 HIV-infected subjects who received 36–52 weeks of indinavir (IDV)/zidovudine (ZDV)/lamivudine (3TC), IDV, or ZDV/3TC. After 1 year of therapy, viral RNA levels in LN of individuals remained detectable but were log
10
= 4 lower than in subjects on the triple drug regimen with interruption of therapy or in those treated with ZDV/3TC alone, who had viral loads in their lymph nodes indistinguishable from those expected for untreated patients. In all cases viral DNA remained detectable in lymph nodes and peripheral blood mononuclear cells (PBMC). When plasma virus suppression was incomplete, lymph node and PBMC cultures were positive and drug resistance developed. These studies indicate that pronounced and sustained suppression of plasma viremia by a potent antiretroviral combination is associated with low HIV RNA levels in the lymph nodes 1 year after treatment. Conversely, the persistence of even modest levels of plasma virus after 1 year of treatment reflects ongoing viral replication, the emergence of drug resistance, and the maintenance of high burdens of virus in the lymph nodes.
Inhibition of interleukin 1β converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage
Authors: Hara Hideaki , Friedlander Robert M. , Gagliardini Valeria , Ayata Cenk , Fink Klaus , Huang Zhihong , Shimizu-Sasamata Masao , Yuan Junying , Moskowitz Michael A.
Published: July 2002

The interleukin 1β converting enzyme (ICE) family plays a pivotal role in programmed cell death and has been implicated in stroke and neurodegenerative diseases. During reperfusion after filamentous middle cerebral artery occlusion, ICE-like cleavage products and tissue immunoreactive interleukin 1β (IL-1β) levels increased in ischemic mouse brain. Ischemic injury decreased after intracerebroventricular injections of ICE-like protease inhibitors,
N
-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.FMK), acetyl-Tyr-Val-Ala-Asp-chloromethylketone, or a relatively selective inhibitor of CPP32-like caspases,
N
-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone, but not a cathepsin B inhibitor,
N
-benzyloxycarbonyl-Phe-Ala-fluoromethylketone. z-VAD.FMK decreased ICE-like cleavage products and tissue immunoreactive IL-1β levels in ischemic mouse brain and reduced tissue damage when administered to rats as well. Only z-VAD.FMK and acetyl-Tyr-Val-Ala-Asp-chloromethylketone reduced brain swelling, and
N
-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone did not attenuate the ischemia-induced increase in tissue IL-1β levels. The three cysteine protease inhibitors significantly improved behavioral deficits, thereby showing that functional recovery of ischemic neuronal tissue can follow blockade of enzymes associated with apoptotic cell death. Finally, we examined the effect of z-VAD.FMK on excitotoxicity and found that it protected against α-amino-3-hydroxy-5-methyl-4-isoxazole propionate-induced or to a lesser extent
N
-methyl-
d
-aspartate-induced excitotoxic brain damage. Thus, ICE-like and CPP32-like caspases contribute to mechanisms of cell death in ischemic and excitotoxic brain injury and provide therapeutic targets for stroke and neurodegenerative brain damage.
Interleukin 8-stimulated phosphatidylinositol-3-kinase activity regulates the migration of human neutrophils independent of extracellular signal-regulated kinase and p38 mitogen-activated protein kinases
Authors: Knall Cindy , Worthen G. Scott , Johnson Gary L.
Published: July 2002

Chemoattractants and chemokines, such as interleukin 8 (IL-8), are defined by their ability to induce directed cell migration of responsive cells. The signal transduction pathway(s) leading to cell migration remain ill defined. We demonstrate that phosphatidylinositol-3-kinase (PI3K) activity, as determined by inhibition using wortmannin and LY294002, is required for IL-8-induced cell migration of human neutrophils. Recently we reported that IL-8 caused the activation of the Ras/Raf/extracellular signal-regulated kinase (ERK) pathway in human neutrophils and that this activation was dependent on PI3K activity. The regulation of cell migration by IL-8 is independent of ERK kinase and ERK activation since the ERK kinase inhibitor PD098059 had no effect on IL-8-induced cell migration of human neutrophils. Additionally, activation of p38-mitogen-activated protein kinase is insufficient and activation of c-Jun N-terminal kinase is unnecessary to induce cell migration of human neutrophils. Therefore, regulation of neutrophil migration appears to be largely independent of the activation of the mitogen-activated protein kinases. The data argue that PI3K activity plays a central role in multiple signal transduction pathways within the human neutrophil leading to distinct cell functions.
Human sulfite oxidase R160Q: Identification of the mutation in a sulfite oxidase-deficient patient and expression and characterization of the mutant enzyme
Authors: Garrett Robert M. , Johnson Jean L. , Graf Tyler N. , Feigenbaum Annette , Rajagopalan K. V.
Published: July 2002

Sulfite oxidase catalyzes the terminal reaction in the degradation of sulfur amino acids. Genetic deficiency of sulfite oxidase results in neurological abnormalities and often leads to death at an early age. The mutation in the sulfite oxidase gene responsible for sulfite oxidase deficiency in a 5-year-old girl was identified by sequence analysis of cDNA obtained from fibroblast mRNA to be a guanine to adenine transition at nucleotide 479 resulting in the amino acid substitution of Arg-160 to Gln. Recombinant protein containing the R160Q mutation was expressed in
Escherichia coli
, purified, and characterized. The mutant protein contained its full complement of molybdenum and heme, but exhibited 2% of native activity under standard assay conditions. Absorption spectroscopy of the isolated molybdenum domains of native sulfite oxidase and of the R160Q mutant showed significant differences in the 480- and 350-nm absorption bands, suggestive of altered geometry at the molybdenum center. Kinetic analysis of the R160Q protein showed an increase in
K
m
for sulfite combined with a decrease in
k
cat
resulting in a decrease of nearly 1,000-fold in the apparent second-order rate constant
k
cat
/
K
m
. Kinetic parameters for the
in vitro
generated R160K mutant were found to be intermediate in value between those of the native protein and the R160Q mutant. Native sulfite oxidase was rapidly inactivated by phenylglyoxal, yielding a modified protein with kinetic parameters mimicking those of the R160Q mutant. It is proposed that Arg-160 attracts the anionic substrate sulfite to the binding site near the molybdenum.
A conserved mode of head segmentation in arthropods revealed by the expression pattern of Hox genes in a spider
Authors: Damen Wim G. M. , Hausdorf Monika , Seyfarth Ernst-August , Tautz Diethard
Published: July 2002

Chelicerates constitute a basic arthropod group with fossil representatives from as early as the Cambrian period. Embryonic development and the subdivision of the segmented body region into a prosoma and an opisthosoma are very similar in all extant chelicerates. The mode of head segmentation, however, has long been controversial. Although all other arthropod groups show a subdivision of the head region into six segments, the chelicerates are thought to have the first antennal segment missing. To examine this problem on a molecular level, we have compared the expression pattern of Hox genes in the spider
Cupiennius salei
with the pattern known from insects. Surprisingly, we find that the anterior expression borders of the Hox genes are in the same register and the same relative segmental position as in
Drosophila
. This contradicts the view that the homologue of the first antennal segment is absent in the spider. Instead, our data suggest that the cheliceral segment is homologous to the first antennal segment and the pedipalpal segment is homologous to the second antennal (or intercalary) segment in arthropods. Our finding implies that chelicerates, myriapods, crustaceans, and insects share a single mode of head segmentation, reinforcing the argument for a monophyletic origin of the arthropods.
Characterization of a murine type II sodium-phosphate cotransporter expressed in mammalian small intestine
Authors: Hilfiker Helene , Hattenhauer Olaf , Traebert Martin , Forster Ian , Murer Heini , Biber Jürg
Published: July 2002

An isoform of the mammalian renal type II Na/P
i
-cotransporter is described. Homology of this isoform to described mammalian and nonmammalian type II cotransporters is between 57 and 75%. Based on major diversities at the C terminus, the new isoform is designed as type IIb Na/P
i
-cotransporter. Na/P
i
-cotransport mediated by the type IIb cotransporter was studied in oocytes of
Xenopus
laevis
. The results indicate that type IIb Na/P
i
-cotransport is electrogenic and in contrast to the renal type II isoform of opposite pH dependence. Expression of type IIb mRNA was detected in various tissues, including small intestine. The type IIb protein was detected as a 108-kDa protein by Western blots using isolated small intestinal brush border membranes and by immunohistochemistry was localized at the luminal membrane of mouse enterocytes. Expression of the type IIb protein in the brush borders of enterocytes and transport characteristics suggest that the described type IIb Na/P
i
-cotransporter represents a candidate for small intestinal apical Na/P
i
-cotransport.
Evidence for a genetic basis for hyperandrogenemia in polycystic ovary syndrome
Authors: Legro Richard S. , Driscoll Deborah , Strauss Jerome F. , Fox Janis , Dunaif Andrea
Published: July 2002

Our preliminary family studies have suggested that some female first-degree relatives of women with polycystic ovary syndrome (PCOS) have hyperandrogenemia
per se
. It was our hypothesis that this may be a genetic trait and thus could represent a phenotype suitable for linkage analysis. To investigate this hypothesis, we examined 115 sisters of 80 probands with PCOS from unrelated families. PCOS was diagnosed by the combination of elevated serum androgen levels and ≤6 menses per year with the exclusion of secondary causes. The sisters were compared with 70 healthy age- and weight-comparable control women with regular menses, no clinical evidence of hyperandrogenemia, and normal glucose tolerance. Twenty-two percent of the sisters fulfilled diagnostic criteria for PCOS. In addition, 24% of the sisters had hyperandrogenemia and regular menstrual cycles. Circulating testosterone (T) and nonsex hormone-binding globulin-bound testosterone (uT) levels in both of these groups of sisters were significantly increased compared with unaffected sisters and control women (
P
< 0.0001 for both T and uT). Probands, sisters with PCOS, and hyperandrogenemic sisters had elevated serum luteinizing hormone levels compared with control women. We conclude that there is familial aggregation of hyperandrogenemia (with or without oligomenorrhea) in PCOS kindreds. In affected sisters, only one-half have oligomenorrhea and hyperandrogenemia characteristic of PCOS, whereas the remaining one-half have hyperandrogenemia
per se
. This familial aggregation of hyperandrogenemia in PCOS kindreds suggests that it is a genetic trait. We propose that hyperandrogenemia be used to assign affected status in linkage studies designed to identify PCOS genes.
A conserved sequence block in murine and human T cell receptor (TCR) Jα region is a composite element that enhances TCR α enhancer activity and binds multiple nuclear factors
Authors: Kuo Chia-Lam , Chen Mei-Ling , Wang Kai , Chou Chuan-Kai , Vernooij Bernard , Seto Donald , Koop Ben F. , Hood Leroy
Published: July 2002

A conserved sequence block (CSB) located in a noncoding region of the mouse and human TCR α/δ loci, showing six differences over 125 nucleotide positions (95% similar), was subjected to detailed analyses in this study. Transient transfection results showed that the CSB-containing element in conjunction with the TCR α enhancer up-regulated the α enhancer activity, whereas no enhancer activity was detected when CSB alone was assayed.
In vitro
occupancy analyses of CSB by nuclear factors reveal the existence of an unexpectedly intricate network of CSB–protein and protein–protein interactions. Lymphoid-specific as well as T-lineage-specific nuclear factors are involved to differentially form CSB-bound complexes in extracts of various tissues and cell lines. Liver was shown to contain factor(s) sequestering thymic CSB-binding factors. Furthermore, the putative binding sites for transcription factors known to be important for lymphoid-lineage development are present in CSB and are targeted by nuclear factors. On the basis of these results, we propose that the CSB element may play a role in shaping the chromatin structure by which the accessibility of TCR α/δ loci to the recombinase complex and/or to the transcriptional apparatus can be controlled.
Heat shock protein 90 mediates macrophage activation by Taxol and bacterial lipopolysaccharide
Authors: Byrd Cynthia A. , Bornmann William , Erdjument-Bromage Hediye , Tempst Paul , Pavletich Nikola , Rosen Neal , Nathan Carl F. , Ding Aihao
Published: July 2002

Taxol, a plant-derived antitumor agent, stabilizes microtubules. Taxol also elicits cell signals in a manner indistinguishable from bacterial lipopolysaccharide (LPS). LPS-like actions of Taxol are controlled by thelpsgene and are independent of binding to the known Taxol target, β-tubulin. Using biotin-labeled Taxol, avidin-agarose affinity chromatography, and peptide mass fingerprinting, we identified two Taxol targets from mouse macrophages and brain as heat shock proteins (Hsps) of the 70- and 90-kDa families. Geldanamycin, a specific inhibitor of the Hsp 90 family, blocked the nuclear translocation of NF-κB and expression of tumor necrosis factor in macrophages treated with Taxol or with LPS. Geldanamycin did not block microtubule bundling by Taxol or macrophage activation by tumor necrosis factor. Thus, Taxol binds Hsps, and Hsp 90 helps mediate the activation of macrophages by Taxol and by LPS.
Translocation of the catalytic domain of diphtheria toxin across planar phospholipid bilayers by its own T domain
Authors: Oh Kyoung Joon , Senzel Lisa , Collier R. John , Finkelstein Alan
Published: July 2002

The T domain of diphtheria toxin is known to participate in the pH-dependent translocation of the catalytic C domain of the toxin across the endosomal membrane, but how it does so, and whether cellular proteins are also required for this process, remain unknown. Here, we report results showing that the T domain alone is capable of translocating the entire C domain across model, planar phospholipid bilayers in the absence of other proteins. The T domain therefore contains the entire molecular machinery for mediating transfer of the catalytic domain of diphtheria toxin across membranes.
Substrate recognition by class I lysyl-tRNA synthetases: A molecular basis for gene displacement
Authors: Ibba Michael , Losey Heather C. , Kawarabayasi Yutaka , Kikuchi Hisasi , Bunjun Shipra , Söll Dieter
Published: July 2002

Lysyl-tRNA synthetases (LysRSs) are unique amongst the aminoacyl-tRNA synthetases in being composed of unrelated class I and class II enzymes. To allow direct comparison between the two types of LysRS, substrate recognition by class I LysRSs was examined. Genes encoding both an archaeal and a bacterial class I enzyme were able to rescue an
Escherichia coli
strain deficient in LysRS, indicating their ability to functionally substitute for a class II LysRS
in vivo
.
In vitro
characterization showed lysine activation and recognition to be tRNA-dependent, an attribute of several class I, but not class II, aminoacyl-tRNA synthetases. Examination of tRNA recognition showed that class I LysRSs recognize the same elements in tRNA
Lys
as their class II counterparts, namely the discriminator base (N73) and the anticodon. This sequence-specific recognition of the same nucleotides in tRNA
Lys
by the two unrelated types of enzyme suggests that tRNA
Lys
predates at least one of the LysRSs in the evolution of the translational apparatus. The only observed variation in recognition was that the G2⋅U71 wobble pair of spirochete tRNA
Lys
acts as antideterminant for class II LysRS but does not alter class I enzyme recognition. This difference in tRNA recognition strongly favors the use of a class I-type enzyme to aminoacylate particular tRNA
Lys
species and provides a molecular basis for the observed displacement of class II by class I LysRSs in certain bacteria.