bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular phylogeny of the extinct ground sloth Mylodon darwinii.
Authors: Höss M , Dilling A , Currant A , Pääbo S
Published: July 2002

DNA was extracted from the remains of 35 ground sloths from various parts of North and South America. Two specimens of Mylodon darwinii, a species that went extinct at the end of the last glaciation, yielded amplifiable DNA. However, of the total DNA extracted, only approximately 1/1000 originated from the sloth, whereas a substantial part of the remainder was of bacterial and fungal origin. In spite of this, > 1100 bp of sloth mitochondrial rDNA sequences could be reconstructed from short amplification products. Phylogenetic analyses using homologous sequences from all extant edentate groups suggest that Mylodon darwinii was more closely related to the two-toed than the three-toed sloths and, thus, that an arboreal life-style has evolved at least twice among sloths. The divergence of Mylodon and the two-toed sloth furthermore allows a date for the radiation of armadillos, anteaters, and sloths to be estimated. This result shows that the edentates differ from other mammalian orders in that they contain lineages that diverged before the end of the Cretaceous Period.
The viruses in all of us: characteristics and biological significance of human endogenous retrovirus sequences.
Authors: Löwer R , Löwer J , Kurth R
Published: July 2002

Human endogenous retroviruses (HERVs) are very likely footprints of ancient germ-cell infections. HERV sequences encompass about 1% of the human genome. HERVs have retained the potential of other retroelements to retrotranspose and thus to change genomic structure and function. The genomes of almost all HERV families are highly defective. Recent progress has allowed the identification of the biologically most active family, HTDV/HERV-K, which codes for viral proteins and particles and is highly expressed in germ-cell tumors. The demonstrable and potential roles of HTDV/HERV-K as well as of other human elements in disease and in maintaining genome plasticity are illustrated.
Correlates of the desired family size among Indian communities.
Authors: Joshi N V , Gadgil M , Patil S
Published: July 2002

The People of India database of the Anthropological Survey of India documents 631 cultural, ecological, and economic traits of the 4635 communities to which the entire Indian population is assigned. Focusing on 1342 communities of South India, we looked for correlates of low (1 or 2 children) and high (4 or more children) desired family size (DFS) reported as the norm for any given community by key informants. We found 10 cultural and 18 economic traits to be significantly correlated to high DFS and 21 cultural and 9 economic traits to low DFS. The economic traits so identified are compatible with high family size being desired by parents who have little capability of investing in quality of offspring, but whose children contribute economically from an early age. In contrast, communities desiring low family size are part of the modern intensive agriculture/organized industry/services sector and invest heavily in educating their children. A composite index based on 27 economic traits (CEI) has a high predictive value with respect to the DFS for the entire set of 4635 Indian communities. The 31 cultural traits highly correlated to high or low DFS constitute 5 clusters that can be identified as characterizing scheduled tribes, scheduled castes, rural and landless lower castes, urban upper castes, and Moslems. Whereas economic traits have similar influence on DFS within each of these ethnic categories, Moslems demonstrate a significantly higher DFS for lower values of CEI.
Glycerol-induced development of catalytically active conformation of Crotalus adamanteus L-amino acid oxidase in vitro.
Authors: Raibekas A A , Massey V
Published: July 2002

The reconstitutable apoprotein of Crotalus adamanteus L-amino acid oxidase was prepared using hydrophobic interaction chromatography. After reconstitution with flavin adenine dinucleotide, the resulting protein was inactive, with a perturbed conformation of the flavin binding site. Subsequently, a series of cosolvent-dependent compact intermediates was identified. The nearly complete activation of the reconstituted apoprotein and the restoration of its native flavin binding site was achieved in the presence of 50% glycerol. We provide evidence that in addition to a merely stabilizing effect of glycerol on native proteins, glycerol can also have a restorative effect on their compact equilibrium intermediates, and we suggest the hydrophobic effect as a dominating force in this in vitro-assisted restorative process.
Cloning and characterization of KAP3: a novel kinesin superfamily-associated protein of KIF3A/3B.
Authors: Yamazaki H , Nakata T , Okada Y , Hirokawa N
Published: July 2002

We previously reported that KIF3A and KIF3B form a heterodimer that functions as a microtubule-based fast anterograde translocator of membranous organelles. We have also shown that this KIF3A/3B forms a complex with other associated polypeptides, named kinesin superfamily-associated protein 3 (KAP3). In the present study, we purified KAP3 protein by immunoprecipitation using anti-KIF3B antibody from mouse testis. Microsequencing was carried out, and we cloned the full-length KAP3 cDNA from a mouse brain cDNA library. Two isoforms of KAP3 exist [KAP3A (793 aa) and KAP3B (772 aa)], generated by alternative splicing in the carboxyl terminus region. Their amino acid sequences have no homology with those of any other known proteins, and prediction of their secondary structure indicated that almost the entire KAP3 molecule is alpha-helical. We produced recombinant KAP3 and KIF3A/3B using a baculovirus-Sf9 expression system. A reconstruction study in Sf9 cells revealed that KAP3 is a globular protein that binds to the tail domain of KIF3A/3B. The immunolocalization pattern of KAP3 was similar to that of KIF3A/3B in nerve cells. In addition, we found that KAP3 does not affect the motor activity of KIF3A/3B. KAP3 was associated with a membrane-bound form of KIF3A/3B in a fractional immunoprecipitation experiment, and since the KIF3 complex was found to bind to membranous organelles in an EM study, KAP3 may regulate membrane binding of the KIF3 complex.
Arabidopsis MBP1 gene encodes a conserved ubiquitin recognition component of the 26S proteasome.
Authors: van Nocker S , Deveraux Q , Rechsteiner M , Vierstra R D
Published: July 2002

Multiubiquitin chain attachment is a key step leading to the selective degradation of abnormal polypeptides and many important regulatory proteins by the eukaryotic 26S proteasome. However, the mechanism by which the 26S complex recognizes this posttranslational modification is unknown. Using synthetic multiubiquitin chains to probe an expression library for interacting proteins, we have isolated an Arabidopsis cDNA, designated MBP1, that encodes a 41-kDa acidic protein exhibiting high affinity for chains, especially those containing four or more ubiquitins. Based on similar physical and immunological properties, multiubiquitin binding affinities, and peptide sequence, MBP1 is homologous to subunit 5a of the human 26S proteasome. Structurally related proteins also exist in yeast, Caenorhabditis, and other plant species. Given their binding properties, association with the 26S proteasome, and widespread distribution, MBP1, S5a, and related proteins likely function as essential ubiquitin recognition components of the 26S proteasome.
Structure of the replication terminus-terminator protein complex as probed by affinity cleavage.
Authors: Pai K S , Bussiere D E , Wang F , White S W , Bastia D
Published: July 2002

The replication terminator protein (RTP) of Bacillus subtilis is a homodimer that binds to each replication terminus and impedes replication fork movement in only one orientation with respect to the replication origin. The three-dimensional structure of the RTP-DNA complex needs to be determined to understand how structurally symmetrical dimers of RTP generate functional asymmetry. The functional unit of each replication terminus of Bacillus subtilis consists of four turns of DNA complexed with two interacting dimers of RTP. Although the crystal structure of the RTP apoprotein dimer has been determined at 2.6-A resolution, the functional unit of the terminus is probably too large and too flexible to lend itself to cocrystallization. We have therefore used an alternative strategy to delineate the three dimensional structure of the RTP-DNA complex by converting the protein into a site-directed chemical nuclease. From the pattern of base-specific cleavage of the terminus DNA by the chemical nuclease, we have mapped the amino acid to base contacts. Using these contacts as distance constraints, with the crystal structure of RTP, we have constructed a model of the DNA-protein complex. The biological implications of the model have been discussed.
Concomitant combination therapy for HIV infection preferable over sequential therapy with 3TC and non-nucleoside reverse transcriptase inhibitors
Authors: Balzarini Jan , Pelemans Heidi , Karlsson Anna , De Clercq Erik , Kleim Jörg-Peter
Published: July 2002

Exposure to 3TC of HIV-1 mutant strains containing non-nucleoside
reverse transcriptase inhibitor (NNRTI)-specific mutations in their
reverse transcriptase (RT) easily selected for double-mutant viruses
that had acquired the characteristic 184-Ile mutation in their RT in
addition to the NNRTI-specific mutations. Conversely, exposure of
3TC-resistant 184-Val mutant HIV-1 strains to nine different NNRTIs
resulted in the rapid emergence of NNRTI-resistant virus strains at a
time that was not more delayed than when wild-type
HIV-1(III
B
) was exposed to the same compounds. The RTs of
these resistant virus strains had acquired the NNRTI-characteristic
mutations in addition to the preexisting 184-Val mutation.
Surprisingly, when the 184-Ile mutant HIV-1 was exposed to a variety of
NNRTIs, the 188-His mutation invariably occurred concomitantly with the
184-Ile mutation in the HIV-1 RT. Breakthrough of this double-mutant
virus was markedly accelerated as compared with the mutant virus
selected from the wild-type or 184-Val mutant HIV-1 strain. The double
(184-Ile + 188-His) mutant virus showed a much more profound resistance
profile against the NNRTIs than the 188-His HIV-1 mutant. In contrast
with the sequential chemotherapy, concomitant combination treatment of
HIV-1-infected cells with 3TC and a variety of NNRTIs resulted in a
dramatic delay of virus breakthrough and resistance development.
Single-channel properties of the nonselective cation conductance induced by neurotensin in dopaminergic neurons
Authors: Chien Pei-Yu , Farkas Ronald H. , Nakajima Shigehiro , Nakajima Yasuko
Published: July 2002

Slow nonselective cation conductances play a central role in
determining the excitability of many neurons, but heretofore this
channel type has not been analyzed at the single-channel level.
Neurotensin (NT) excites cultured dopaminergic neurons from the ventral
tegmental area primarily by increasing such a cation conductance. Using
the outside–out configuration of the patch clamp, we elicited
single-channel activity of this NT-induced cation channel. Channel
activity was blocked by the nonpeptide NT antagonist SR48692,
indicating that the response was mediated by NT receptors. The channel
opened in both solitary form and in bursts. The reversal potential was
−4.2 ± 1.7 mV, and the elementary conductance was 31 pS at −67
mV with [Na
+
]
o
= 140 mM,
[Cs
+
]
o
= 5 mM,
[Na
+
]
i
= 88 mM, and
[Cs
+
]
i
= 74 mM. Thus, the channel was
permeable to both Na
+
and Cs
+
. From these
characteristics, it is likely that this channel is responsible for the
whole-cell current we studied previously. In guanosine
5′-[γ-thio]triphosphate-loaded cells, NT irreversibly activated
about half of the channel activity, suggesting that at least part of
the response was mediated by a G protein. Similar channel activity
could be induced occasionally in the cell-attached configuration by
applying NT outside the patch region.
Converting cancer genes into killer genes.
Authors: Da Costa L T , Jen J , He T C , Chan T A , Kinzler K W , Vogelstein B
Published: July 2002

Over the past decade, it has become clear that tumorigenesis is driven by alterations in genes that control cell growth or cell death. Theoretically, the proteins encoded by these genes provide excellent targets for new therapeutic agents. Here, we describe a gene therapy approach to specifically kill tumor cells expressing such oncoproteins. In outline, the target oncoprotein binds to exogenously introduced gene products, resulting in transcriptional activation of a toxic gene. As an example, we show that this approach can be used to specifically kill cells overexpressing a mutant p53 gene in cell culture. The strategy may be generally applicable to neoplastic diseases in which the underlying patterns of genetic alterations or abnormal gene expression are known.
DNA sequences of Alu elements indicate a recent replacement of the human autosomal genetic complement.
Authors: Knight A , Batzer M A , Stoneking M , Tiwari H K , Scheer W D , Herrera R J , Deininger P L
Published: July 2002

DNA sequences of neutral nuclear autosomal loci, compared across diverse human populations, provide a previously untapped perspective into the mode and tempo of the emergence of modern humans and a critical comparison with published clonally inherited mitochondrial DNA and Y chromosome measurements of human diversity. We obtained over 55 kilobases of sequence from three autosomal loci encompassing Alu repeats for representatives of diverse human populations as well as orthologous sequences for other hominoid species at one of these loci. Nucleotide diversity was exceedingly low. Most individuals and populations were identical. Only a single nucleotide difference distinguished presumed ancestral alleles from descendants. These results differ from those expected if alleles from divergent archaic populations were maintained through multiregional continuity. The observed virtual lack of sequence polymorphism is the signature of a recent single origin for modern humans, with general replacement of archaic populations.
An essential cell division gene of
Drosophila
, absent from
Saccharomyces
, encodes an unusual protein with tubulin-like and myosin-like peptide motifs
Authors: Miklos George L. Gabor , Yamamoto M.-T. , Burns R. G. , Maleszka R.
Published: July 2002

Null mutations at the
misato
locus of
Drosophila melanogaster
are associated with irregular chromosomal segregation at cell division. The consequences for morphogenesis are that mutant larvae are almost devoid of imaginal disk tissue, have a reduction in brain size, and die before the late third-instar larval stage. To analyze these findings, we isolated cDNAs in and around the
misato
locus, mapped the breakpoints of chromosomal deficiencies, determined which transcript corresponded to the
misato
gene, rescued the cell division defects in transgenic organisms, and sequenced the genomic DNA. Database searches revealed that
misato
codes for a novel protein, the N-terminal half of which contains a mixture of peptide motifs found in α-, β-, and γ-tubulins, as well as a motif related to part of the myosin heavy chain proteins. The sequence characteristics of
misato
indicate either that it arose from an ancestral tubulin-like gene, different parts of which underwent convergent evolution to resemble motifs in the conventional tubulins, or that it arose by the capture of motifs from different tubulin genes. The
Saccharomyces cerevisiae
genome lacks a true homolog of the
misato
gene, and this finding highlights the emerging problem of assigning functional attributes to orphan genes that occur only in some evolutionary lineages.
Homodimerization of the human interleukin 4 receptor α chain induces Cɛ germline transcripts in B cells in the absence of the interleukin 2 receptor γ chain
Authors: Fujiwara Hiroshi , Hanissian Silva H. , Tsytsykova Alla , Geha Raif S.
Published: July 2002

The cytokines interleukin (IL)-4 and IL-13 play a critical role in inducing Cɛ germline transcripts and IgE isotype switching in human B cells. The IL-4 receptor (IL-4R) in B cells is composed of two chains, the IL-4-binding IL-4Rα chain, which is shared with the IL-13R, and the IL-2Rγ (γc) chain, which is shared with IL-7R, IL-9R, and IL-15R. IL-4 induces Cɛ germline transcripts and IgE isotype switching in B cells from patients with γc chain deficiency. Induction of Cɛ germline transcripts by IL-4 in B cells that lack the γc chain may involve signaling via the IL-13R. Alternatively, the IL-4Rα chain may transduce intracellular signals that lead to Cɛ gene transcription independently of its association with other chains. We show that ligand-induced homodimerization of chimeric surface receptors consisting of the extracellular and transmembrane domains of the erythropoietin receptor and of the intracellular domain of IL-4Rα induces Janus kinase 1 (Jak1) activation, STAT6 activation, and Cɛ germline transcripts in human B cell line BJAB. Disruption of the Jak1-binding proline-rich Box1 region of IL-4Rα abolished signaling by this chimeric receptor. Furthermore, B cells transfected with a chimeric CD8α/IL-4Rα receptor, which is expressed on the cell surface as a homodimer, constitutively expressed Cɛ germline transcripts. These results suggest that homodimerization of the IL-4Rα chain is sufficient to transduce Jak1-dependent intracellular signals that lead to IgE isotype switching.
Expression of HLA class I-specific inhibitory natural killer cell receptors in HIV-specific cytolytic T lymphocytes: Impairment of specific cytolytic functions
Authors: De Maria Andrea , Ferraris Anna , Guastella Marina , Pilia Simonetta , Cantoni Claudia , Polero Laura , Mingari Maria Cristina , Bassetti Dante , Fauci Anthony S. , Moretta Lorenzo
Published: July 2002

Human T lymphocytes have been shown to express inhibitory natural killer cell receptors (NKR), which can down-regulate T cell antigen receptor-mediated T cell function, including cytolytic activity. In the present study, we demonstrate that CD3+NKR+cells can be identified in HIV-infected patients. HIV-specific cytolytic activity was analyzed in five patients in whom autologous lymphoblastoid B cell lines could be derived as a source of autologous target cells. Phytohemagglutinin-activated T cell populations that had been cultured in interleukin 2 displayed HIV-specific cytotoxic T lymphocyte (CTL) activity against HIV env, gag, pol, and nef in 3 of 5 patients. Addition of anti-NKR mAb of IgM isotype could increase the specific CTL activity. Moreover, in one additional patient, HIV-specific CTL activity was undetectable; however, after addition of anti-NKR mAb such CTL activity appearedde novo. Similar results were obtained by analysis of CD3+NKR+clones derived from two patients. These data provide direct evidence that CD3+NKR+cells may include antigen (HIV)-specific CTLs and that mAb-mediated masking of inhibitory NKR may revert the down-regulation of CTL function.
Extensive purification of a putative RNA polymerase I holoenzyme from plants that accurately initiates rRNA gene transcription
in vitro
Authors: Saez-Vasquez Julio , Pikaard Craig S.
Published: July 2002

RNA polymerase I (pol I) is a nuclear enzyme whose function is to transcribe the duplicated genes encoding the precursor of the three largest ribosomal RNAs. We report a cell-free system from broccoli (
Brassica oleracea
) inflorescence that supports promoter-dependent RNA pol I transcription
in vitro
. The transcription system was purified extensively by DEAE-Sepharose, Biorex 70, Sephacryl S300, and Mono Q chromatography. Activities required for pre-rRNA transcription copurified with the polymerase on all four columns, suggesting their association as a complex. Purified fractions programmed transcription initiation from the
in vivo
start site and utilized the same core promoter sequences required
in vivo
. The complex was not dissociated in 800 mM KCl and had a molecular mass of nearly 2 MDa based on gel filtration chromatography. The most highly purified fractions contain ≈30 polypeptides, two of which were identified immunologically as RNA polymerase subunits. These data suggest that the occurrence of a holoenzyme complex is probably not unique to the pol II system but may be a general feature of eukaryotic nuclear polymerases.
Synapsin I interacts with c-Src and stimulates its tyrosine kinase activity
Authors: Onofri Franco , Giovedì Silvia , Vaccaro Paola , Czernik Andrew J. , Valtorta Flavia , De Camilli Pietro , Greengard Paul , Benfenati Fabio
Published: July 2002

Synapsin I is a synaptic vesicle-associated phosphoprotein that has been implicated in the formation of presynaptic specializations and in the regulation of neurotransmitter release. The nonreceptor tyrosine kinase c-Src is enriched on synaptic vesicles, where it accounts for most of the vesicle-associated tyrosine kinase activity. Using overlay, affinity chromatography, and coprecipitation assays, we have now shown that synapsin I is the major binding protein for the Src homology 3 (SH3) domain of c-Src in highly purified synaptic vesicle preparations. The interaction was mediated by the proline-rich domain D of synapsin I and was not significantly affected by stoichiometric phosphorylation of synapsin I at any of the known regulatory sites. The interaction of purified c-Src and synapsin I resulted in a severalfold stimulation of tyrosine kinase activity and was antagonized by the purified c-Src-SH3 domain. Depletion of synapsin I from purified synaptic vesicles resulted in a decrease of endogenous tyrosine kinase activity. Portions of the total cellular pools of synapsin I and Src were coprecipitated from detergent extracts of rat brain synaptosomal fractions using antibodies to either protein species. The interaction between synapsin I and c-Src, as well as the synapsin I-induced stimulation of tyrosine kinase activity, may be physiologically important in signal transduction and in the modulation of the function of axon terminals, both during synaptogenesis and at mature synapses.
Dual activity maps in primate visual cortex produced by different temporal patterns of
zif268
mRNA and protein expression
Authors: Chaudhuri Avi , Nissanov Jonathan , Larocque Sylvie , Rioux Lise
Published: July 2002

The inducible nature of the immediate-early genes (IEGs) c-
fos
and
zif268
allows their products to be used as activity markers in the brain. The utility of such markers in general is restricted because they can resolve only neurons activated by a single stimulus. To overcome this limitation, we have developed a double-label technique that exploits the dissimilar time course of
zif268
mRNA and protein induction, allowing them to be separately induced by two different stimuli and independently stained to provide a visual display of neurons that are responsive to each stimulus. Two powerful features of this new imaging technique—the possibility of staining separate populations of activated neurons and the ability to visualize them at the cellular level—should extend IEG applications in biological activity mapping.
Defining the chemical groups essential for
Tetrahymena
group I intron function by nucleotide analog interference mapping
Authors: Strobel Scott A. , Shetty Kanaka
Published: July 2002

Improved atomic resolution biochemical methods are needed to identify the chemical groups within an RNA that are essential to its activity. As a step toward this goal, we report the use of 5′-
O
-(1-thio)inosine monophosphate (IMPαS) in a nucleotide analog interference mapping (NAIM) assay that makes it possible to simultaneously, yet individually, determine the contribution of almost every N2 exocyclic amine of G within a large RNA. Using IMPαS, we identified the exocyclic amines that are essential for 5′ or 3′ exon ligation by the
Tetrahymena
group I intron. We report that the amino groups of three phylogenetically conserved guanosines (G111, G112, and G303) are important for 3′ exon ligation. The amine of G22, as well as the amines of the other four guanosines within the P1 helix, are essential for ligation of the 5′ exon. Previous work has shown that point mutation of either G22 or G303 to an adenosine (A) substantially reduces activity. Like inosine, adenosine lacks an N2 amino group. Interference rescue of the G22A and G303A point mutations was detected at the site of mutation by NAIM using 5′-
O
-(1-thio)diaminopurine riboside monophosphate (DMPαS), an adenosine analog that has an N2 exocyclic amine. The G22A point mutant could also be rescued by incorporation of DMPαS at A24. By analogy to genetics, there are interference phenotypes comparable to loss of function, reversion, and suppression. This method can be readily extended to other nucleotide analogs for the analysis of chemical groups essential to a variety of RNA and DNA activities.
Structural basis for efficient phosphorylation of 3′-azidothymidine monophosphate by
Escherichia coli
thymidylate kinase
Authors: Lavie Arnon , Ostermann Nils , Brundiers Ralf , Goody Roger S. , Reinstein Jochen , Konrad Manfred , Schlichting Ilme
Published: July 2002

The crystal structures of
Escherichia coli
thymidylate kinase (TmpK) in complex with
P
1
-(5′-adenosyl)-
P
5
-(5′-thymidyl)pentaphosphate and
P
1
-(5′-adenosyl)P
5
-[5′-(3′-azido-3′-deoxythymidine)] pentaphosphate have been solved to 2.0-Å and 2.2-Å resolution, respectively. The overall structure of the bacterial TmpK is very similar to that of yeast TmpK. In contrast to the human and yeast TmpKs, which phosphorylate 3′-azido-3′-deoxythymidine 5′-monophosphate (AZT-MP) at a 200-fold reduced turnover number (
k
cat
) in comparison to the physiological substrate dTMP, reduction of
k
cat
is only 2-fold for the bacterial enzyme. The different kinetic properties toward AZT-MP between the eukaryotic TmpKs and
E. coli
TmpK can be rationalized by the different ways in which these enzymes stabilize the presumed transition state and the different manner in which a carboxylic acid side chain in the P loop interacts with the deoxyribose of the monophosphate. Yeast TmpK interacts with the 3′-hydroxyl of dTMP through Asp-14 of the P loop in a bidentate manner: binding of AZT-MP results in a shift of the P loop to accommodate the larger substituent. In
E. coli
TmpK, the corresponding residue is Glu-12, and it interacts in a side-on fashion with the 3′-hydroxyl of dTMP. This different mode of interaction between the P loop carboxylic acid with the 3′ substituent of the monophosphate deoxyribose allows the accommodation of an azido group in the case of the
E. coli
enzyme without significant P loop movement. In addition, although the yeast enzyme uses Arg-15 (a glycine in
E. coli
) to stabilize the transition state,
E. coli
seems to use Arg-153 from a region termed Lid instead. Thus, the binding of AZT-MP to the yeast TmpK results in the shift of a catalytic residue, which is not the case for the bacterial kinase.
Telomerase expression in chickens: Constitutive activity in somatic tissues and down-regulation in culture
Authors: Venkatesan Ranga N. , Price Carolyn
Published: July 2002

Although human and rodent telomeres have been studied extensively, very little is known about telomere dynamics in other vertebrates. Moreover, our current dependence on mice as a model for human tumorigenesis and aging poses a problem because human and mouse telomere biology is very different. To explore whether chickens might provide a more useful model, we have examined telomerase activity and telomere length in chicken tissues as well as in primary cell cultures. Although chicken telomeres resemble human telomeres in that they are 8–20 kb in length, the distribution of telomerase activity in chickens resembles what is found in mice. Active enzyme is present in germline tissue as well as in a wide range of somatic tissues. Because chicken cells exhibit extremely low rates of spontaneous immortalization, this finding indicates that constitutive telomerase expression does not necessarily lead to an increased immortalization frequency. Finally, we found that telomerase activity is greatly down-regulated when primary cultures are established from chicken embryos. Although this down-regulation explains the telomere loss and replicative senescence that we observed in fibroblast cultures, it raises questions concerning how relevant studies of senescence in primary cell cultures are to aging in whole animals.
Genes in the pX region of human T cell leukemia virus I influence Vav phosphorylation in T cells
Authors: Mahana W. , Zhao T. M. , Teller R. , Robinson M. A. , Kindt T. J.
Published: July 2002

Human T cell leukemia virus I (HTLV-I) causes acute leukemic disease in a low percentage of infected individuals through obscure mechanisms. Our studies compare two rabbit HTLV-I-infected T cell lines: one, RH/K34, causes lethal experimental leukemia and the other, RH/K30, mediates asymptomatic infection. We show herein that the product of the protooncogenevavis constitutively Tyr-phosphorylated in RH/K34 but not in RH/K30. A role for the retrovirus in phosphorylation of Vav was assigned by transfection experiments with molecular clones of HTLV-I derived from the two lines. The HTLV-I molecular clone from RH/K30, but not that from RH/K34, down-regulates Vav phosphorylation in a Herpesvirus ateles-transformed T cell line. Use of recombinant virus clones revealed that a pX region sequence differing by two nucleotides between the two clones mediates this down-regulation. Because Vav is involved in T cell signaling and Vav phosphorylation occurs upon activation of T cells, control of the activation state of Vav by viral proteins may relate to the leukemogenic potential of certain HTLV-I-infected cells.
Transcriptional sequencing: A method for DNA sequencing using RNA polymerase
Authors: Sasaki Nobuya , Izawa Masaki , Watahiki Masanori , Ozawa Kaori , Tanaka Takumi , Yoneda Yuko , Matsuura Shuji , Carninci Piero , Muramatsu Masami , Okazaki Yasushi , Hayashizaki Yoshihide
Published: July 2002

We have developed a sequencing method based on the RNA polymerase chain termination reaction with rhodamine dye attached to 3′-deoxynucleoside triphosphate (3′-dNTP). This method enables us to conduct a rapid isothermal sequencing reaction in <30 min, to reduce the amount of template required, and to do PCR direct sequencing without the elimination of primers and 2′-dNTP, which disturbs the Sanger sequencing reaction. An accurate and longer read length was made possible by newly designed four-color dye-3′-dNTPs and mutated RNA polymerase with an improved incorporation rate of 3′-dNTP. This method should be useful for large-scale sequencing in genome projects and clinical diagnosis.
Virus-inducible expression of a host chemokine gene relies on replication-linked mRNA stabilization
Authors: Koga Takeharu , Sardiña Evelio , Tidwell Rose M. , Pelletier Mark , Look Dwight C. , Holtzman Michael J.
Published: July 2002

Analysis of the host response to viral infection generally has focused on the capacity of viruses to activate or repress transcription of cellular genes, and this approach is also characteristic of work on RNA viruses such as respiratory syncytial virus (RSV). In the present study, it appeared initially that RSV-driven expression of a critical immune regulator, the β-chemokine RANTES (regulated upon activation, normal T cell expressed and secreted), in primary-culture airway epithelial cells also depended on inducible gene transcription because expression was accompanied by coordinate increases in transcriptional initiation rate and gene promoter activity. However, RSV-driven increases in RANTES gene transcription and promoter activity were small and transient relative to RANTES expression, and they were no different in size and duration than for inactivated RSV that was incapable of fully inducing RANTES expression. These findings suggested that the increase in RANTES gene transcription was not sufficient for inducible expression and that critical regulatory effects occurred at a posttranscriptional level. This type of mechanism for virus-inducible expression of RANTES was established when we found that replicating (but not inactivated) RSV markedly increased RANTES mRNA half-life (from 0.8 to 6.8 h). In addition, RNase protection assays of heterologous promoter/reporter plasmids indicate that basal instability of RANTES mRNA is mediated at least in part by nucleotides 11–389 of the RANTES gene, and this region is also the target for induction by virus. The distinct pathway for production of RANTES (in combination with cytokine-dependent expression of RANTES and related immune-response genes) may more effectively coordinate immune cell interaction with epithelial barrier cells to mediate host defense.
Ligation of the CD4 receptor induces activation-independent down-regulation ofl-selectin
Authors: Marschner Susanne , Freiberg Benjamin A. , Kupfer Abraham , Hünig Thomas , Finkel Terri H.
Published: July 2002

Lymphocyte circulation plays an important role in the generation of a specific immune response. Mature lymphocytes continuously circulate between blood and lymph, entering the lymphoid tissue via high endothelial venules. Trafficking across high endothelial venules of peripheral lymph nodes (PLN) depends on the expression ofl-selectin. It has been shown thatl-selectin is rapidly cleaved from the surface by a metalloproteinase afterin vitroactivation. Here, we show that ligation of CD4, without ligation of the T cell receptor for antigen, causes down-regulation ofl-selectin on T helper cells. This down-regulation is caused by proteolytic cleavage by a metalloproteinase and is reversible by the addition of hydroxamic acid-based metalloproteinase inhibitors. We show thatin vivodown-regulation ofl-selectin in huCD4tg mice by mAb reduces the homing of lymphocytes to PLN in adoptive transfer experiments. Because CD4 is a coreceptor for HIV-1, the down-regulation ofl-selectin induced by CD4 ligation could play a role in the pathogenesis of AIDS. We provide evidence that CD4 ligation by HIV-1 induces metalloproteinase-dependentl-selectin down-regulation. Reduced levels ofl-selectin expression might contribute to immune deficiency in individuals infected with HIV by inhibiting T cell redistribution and decreasing the probability of an encounter between specific lymphocytes and viral antigens in PLN.
Further examination of the
Xist
promoter-switch hypothesis in X inactivation: Evidence against the existence and function of a P
0
promoter
Authors: Warshawsky David , Stavropoulos Nicholas , Lee Jeannie T.
Published: July 2002

The onset of X inactivation coincides with accumulation of
Xist
RNA along the future inactive X chromosome. A recent hypothesis proposed that accumulation is initiated by a promoter switch within
Xist.
In this hypothesis, an upstream promoter (P
0
) produces an unstable transcript, while the known downstream promoter (P
1
) produces a stable RNA. To test this hypothesis, we examined expression and half-life of
Xist
RNA produced from an
Xist
transgene lacking P
0
but retaining P
1
. We confirm the previous finding that P
0
is dispensable for
Xist
expression in undifferentiated cells and that P
1
can be used in both undifferentiated and differentiated cells. Herein, we show that
Xist
RNA initiated at P
1
is unstable and does not accumulate. Further analysis indicates that the transcriptional boundary at P
0
does not represent the 5′ end of a distinct
Xist
isoform. Instead, P
0
is an artifact of cross-amplification caused by a pseudogene of the highly expressed ribosomal protein S12 gene
Rps12
. Using strand-specific techniques, we find that transcription upstream of P
1
originates from the DNA strand opposite
Xist
and represents the 3′ end of the antisense
Tsix
RNA. Thus, these data do not support the existence of a P
0
promoter and suggest that mechanisms other than switching of functionally distinct promoters control the up-regulation of
Xist
.
Genetic dissection of protein–protein interactions in multi-tRNA synthetase complex
Authors: Rho Seung Bae , Kim Min Jung , Lee Jong Sang , Seol Wongi , Motegi Hiromi , Kim Sunghoon , Shiba Kiyotaka
Published: July 2002

Cytoplasmic aminoacyl-tRNA synthetases of higher eukaryotes acquired extra peptides in the course of their evolution. It has been thought that these appendices are related to the occurrence of the multiprotein complex consisting of at least eight different tRNA synthetase polypeptides. This complex is believed to be a signature feature of metazoans. In this study, we used multiple sequence alignments to infer the locations of the peptide appendices from human cytoplasmic tRNA synthetases found in the multisynthetase complex. The selected peptide appendices ranged from 22 aa of aspartyl-tRNA synthetase to 267 aa of methionyl-tRNA synthetase. We then made genetic constructions to investigate interactions between all 64 combinations of these peptides that were individually fused to nonsynthetase test proteins. The analyses identified 11 (10 heterologous and 1 homologous) interactions. The six peptide-dependent interactions paralleled what had been detected by crosslinking methods applied to the isolated multisynthetase complex. Thus, small peptide appendices seem to link together different synthetases into a complex. In addition, five interacting pairs that had not been detected previously were suggested from the observed peptide-dependent complexes.
A first trial of retrospective collaboration for positional cloning in complex inheritance: Assay of the cytokine region on chromosome 5 by the Consortium on Asthma Genetics (COAG)
Authors: Lonjou C. , Barnes K. , Chen H. , Cookson W. O. C. M. , Deichmann K. A. , Hall I. P. , Holloway J. W. , Laitinen T. , Palmer L. J. , Wjst M. , Morton N. E.
Published: July 2002

The central problem of complex
inheritance is to map oligogenes for disease susceptibility,
integrating linkage and association over samples that differ in several
ways. Combination of evidence over multiple samples with 1,037 families
supports loci contributing to asthma susceptibility in the cytokine
region on 5q [maximum logarithm of odds (lod) = 2.61 near IL-4],
but no evidence for atopy. The principal problems with retrospective
collaboration on linkage appear to have been solved, providing far more
information than a single study. A multipoint lod table evaluated at
commonly agreed reference loci is required for both collaboration and
metaanalysis, but variations in ascertainment, pedigree structure,
phenotype definition, and marker selection are tolerated. These methods
are invariant with statistical methods that increase the power of lods
and are applicable to all diseases, motivating collaboration rather
than competition. In contrast to linkage, positional cloning by allelic
association has yet to be extended to multiple samples, a prerequisite
for efficient combination with linkage and the greatest current
challenge to genetic epidemiology.
A lower jaw of
Pondaungia cotteri
from the Late Middle Eocene Pondaung Formation (Myanmar) confirms its anthropoid status
Authors: Chaimanee Yaowalak , Thein Tin , Ducrocq Stéphane , Soe Aung Naing , Benammi Mouloud , Tun Than , Lwin Thit , Wai San , Jaeger Jean-Jacques
Published: July 2002

Pondaungia cotteri
is the largest primate known from the Late Middle Eocene Pondaung Formation, Myanmar. Its taxonomic status has been the subject of much debate because of the fragmentary nature of its remains. Initially described as an anthropoid, some authors recently have associated it with adapid primates. These debates have been fueled not only by the incompleteness of the fossils attributed to
Pondaungia
but also by the reticence of many authors to regard Asia as an important evolutionary theater for Eocene anthropoids. During the November 1998 Myanmar-French Pondaung Expedition, a right lower jaw was discovered that yields the most nearly complete dentition of
Pondaungia cotteri
ever found: it shows the complete horizontal ramus, alveoli for the second incisor and canine, three premolars, and three molars. The symphysis showed all characteristics of anthropoids but was unfused. The canine root is large, the first premolar is absent, and the second premolar is single-rooted, reduced, and oblique in the tooth row, as in anthropoids. The premolars show a reduced mesio-distal length compared with the tooth row, and their morphology is very similar to that of
Amphipithecus mogaungensis
. Therefore, the two Pondaung taxa appear to be closely related to each other, with
Siamopithecus
as their sister taxon.
Similar regions of human chromosome 3 are eliminated from or retained in human/human and human/mouse microcell hybrids during tumor growth in severe combined immunodeficient (SCID) mice
Authors: Yang Ying , Kost-Alimova Maria , Ingvarsson Sigurdur , Qianhui Qiu , Kiss Hajnalka , Szeles Anna , Kholodnyuk Irina , Cuthbert Andrew , Klein George , Imreh Stephan
Published: July 2002

By passaging microcell hybrids (MCHs) containing human chromosome 3
(chr3) on A9 mouse fibrosarcoma background through severe combined
immunodeficient (SCID) mice (elimination test), we have previously
defined a 1-Mb-long common eliminated region 1 (CER1) at 3p21.3, a
second eliminated region (ER2) at 3p21.1-p14 and a common retained
region (CRR) at 3q26-qter. In the present work, chr3 was transferred by
microcell fusion into the human nonpapillary renal cell carcinoma line
KH39 that contained uniparentally disomic chr3. Four MCHs were
generated. Compared with KH39, they developed fewer and smaller tumors,
which grew after longer latency periods in SCID mice. The tumors were
analyzed in comparison with corresponding MCHs by chr3 arm-specific
painting, 19 fluorescent
in situ
hybridization (FISH)
probes, and 27 polymorphic markers. Three MCHs that maintained the
intact exogenous chr3
in vitro
lost one 3p copy in all
11 tumors. Seven of 11 tumors lost the exogenous 3p, whereas four
tumors contained mixed cell populations that lacked either the
exogenous or one endogenous KH39 derived 3p. In one MCH the
exogenous chr3 showed deletions within CER1 and ER2 already
in
vitro
. It remained essentially unchanged in 8/9 derived
tumors. The third, exogenous copy of the 3q26–q27 region (part of CRR)
was retained in 16/20 tumors. It can be concluded that the
human/human MCH-based elimination test identifies similar eliminated
and retained regions on chr3 as the human/murine MCH-based test.
Culture and perceptions of self—other similarity
Authors: Satterwhite Robert C. , Feldman Jack M. , Catrambone Richard , Dai Liang‐Yu
Published: July 2002

In testing possible cultural effects of the use of the self as an habitual reference point to which others are compared, we expected that: (a) individualistic participants (i.e., those who give priority to personal goals) would rate self—other similarity higher when asked “How similar is X to you?” than when asked “How similar are you to X?”, whereas nondirectional similarity judgements (“How similar are these two people?”) would resemble the former directional comparison; (b) collectivistic participants (i.e., those who give priority to in‐group goals) would show a weaker or, possibly, reversed pattern, especially using in‐group comparison others. Neither hypothesis was upheld. However, the individualists perceived the in‐group to be relatively more similar to themselves as compared to the collectivists. This difference cannot be explained by response bias, status asymmetry, or role differentiation. We propose an explanation in terms of the differential relationship between self and other representations for people from collectivist versus individualist cultures.
Item Interference and Time Delays in Working Memory: Immediate Serial Recall
Authors: Dosher Barbara Anne
Published: July 2002

Limitations on immediate recall span have been related by some to temporal limitations of an articulatory rehearsal loop (Baddeley, 1986), and by others to retroactive interference from subsequent items (Lewandowsky & Murdock, 1989; Neath & Nairne, 1995). In the current study, the number of intervening items and retention delays were partially decoupled by varying presentation rate and materials sets. Recently, memory span effects often attributed to temporal limits in rehearsal were shown to be directly related to the output delays during the act of recall (Dosher & Ma, 1998). A functional model of working memory provides a framework within which to evaluate the relative contributions of item interference and retention delays in the accuracy of recall at each serial position. Output delays during recall and interference from items during study were the primary factors in limiting recall, with small additional effects of time delays during study.
GCSE mathematics in further education: challenging beliefs and practices
Authors: Swan Malcolm
Published: July 2002

This paper describes the outcomes of a two‐year study on the beliefs and practices adopted by four FE teachers in their attempt to teach one‐year GCSE retake classes and the subsequent effects on students' learning. In the second year of the study, an attempt was made to perturb these practices by introducing a substantial collection of classroom discussion resources. These focused on well‐known conceptual obstacles and were intended to provoke cognitive conflict and reflection. Modest, yet significant learning gains were observed with two of the classes, but not with the others. Reasons for this appear to be related to teachers' beliefs about learning.
The role of powerful pedagogical strategies in curriculum development
Authors: Leat David , Higgins Steve
Published: July 2002

Despite the frenetic pace of curriculum change in England and Wales it is highly questionable to what extent classrooms have changed since the inception of the National Curriculum, as little attention has been paid to teacher development. This article describes a human‐scale approach to professional and curriculum development that relies upon powerful pedagogical strategies. The characteristics and popularity of these strategies are described. The model developed in the study of Effective Teachers of Numeracy (Askew et al., 1997) is used to explain how the use of the strategies can lead to professional and curriculum development. Particular attention is paid, through the reflective accounts of teachers who have used the strategies, to the role of pupil response in accelerating change. Finally the article discusses the way in which the strategies accord with much of what is known about how teachers plan.
Alcohol and heart muscle disease
Authors: FERNÁNDEZ‐SOLÀ JOAQUIM , ESTRUCH RAMON , URBANO‐MARQUEZ ALVARO
Published: July 2002

Abstract Ethanol consumption may induce acute and chronic effects on the myocardium. High‐dose acute ethanol intake may induce a decrease in myocardial contraction and produce a variety of rhythm disturbances. These effects are more relevant in patients with underlying cardiomyopathy. Chronic ethanol intake may induce the development of a dilated cardiomyopathy, which is clinically and functionally similar to idiopathic dilated cardiomyopathy. Alcoholic cardiomyopathy is potentially reversible with abstinence. The prognosis depends on the persistence or abstinence of ethanol intake. There is a positive correlation between alcoholic cardiomyopathy and the presence of other ethanol‐related diseases, such as skeletal myopathy and cirrhosis. In patients with a specific ethanol‐related disease, the possible presence of other complications of alcoholism should be ruled out. Although there are several factors potentially implicated in the pathogenesis of alcohol‐related myocardial damage, ethanol itself may induce direct myocardial lesions, which are dose‐related and independent of nutrition, protein or ionic deficiencies. The most relevant pathogenic studies on alcoholic cardiomyopathy are based on the disruption of membrane permeability and ionic fluxes mediated by ethanol, inducing a decrease in the calcium transients through the sarcolemma and interfering with the excitation‐contraction coupling of myocytes. Cell energy depletion or protein‐turnover disruption may contribute to the deleterious effect of ethanol on the myocardium.