bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning and functional analysis of SUT-1, a sulfate transporter from human high endothelial venules
Authors: Girard Jean-Philippe , Baekkevold Espen S. , Feliu Jacques , Brandtzaeg Per , Amalric François
Published: July 2002

High endothelial venules (HEV) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. One of the major characteristics of HEV endothelial cells (HEVEC) is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. Here, we show that HEVEC express two functional classes of sulfate transporters defined by their differential sensitivity to the anion-exchanger inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), and we report the molecular characterization of a DIDS-resistant sulfate transporter from human HEVEC, designated SUT-1. SUT-1 belongs to the family of Na
+
-coupled anion transporters and exhibits 40–50% amino acid identity with the rat renal Na
+
/sulfate cotransporter, NaSi-1, as well as with the human and rat Na
+
/dicarboxylate cotransporters, NaDC-1/SDCT1 and NaDC-3/SDCT2. Functional expression studies in cRNA-injected
Xenopus
laevis
oocytes showed that SUT-1 mediates high levels of Na
+
-dependent sulfate transport, which is resistant to DIDS inhibition. The
SUT-1
gene mapped to human chromosome 7q33. Northern blotting analysis revealed that SUT-1 exhibits a highly restricted tissue distribution, with abundant expression in placenta. Reverse transcription–PCR analysis indicated that SUT-1 and the diastrophic dysplasia sulfate transporter (DTD), one of the two known human DIDS-sensitive sulfate transporters, are coexpressed in HEVEC. SUT-1 and DTD could correspond, respectively, to the DIDS-resistant and DIDS-sensitive components of sulfate uptake in HEVEC. Together, these results demonstrate that SUT-1 is a distinct human Na
+
-coupled sulfate transporter, likely to play a major role in sulfate incorporation in HEV.
Probing the
Saccharomyces cerevisiae
centromeric DNA (
CEN
DNA)–binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy
Authors: Pietrasanta Lía I. , Thrower Douglas , Hsieh Wan , Rao Shashirekha , Stemmann Olaf , Lechner Johannes , Carbon John , Hansma Helen
Published: July 2002

Yeast centromeric DNA (
CEN
DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in
CEN
DNA. The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore. We have examined the structure of the CBF3–
CEN
DNA complex by atomic force microscopy. Assembly of CBF3–
CEN
DNA complexes was performed by combining purified CBF3 proteins with a DNA fragment that includes the
CEN
region from yeast chromosome III. Atomic force microscopy images showed DNA molecules with attached globular bodies. The contour length of the DNA containing the complex is ≈9% shorter than the DNA alone, suggesting some winding of DNA within the complex. The measured location of the single binding site indicates that the complex is located asymmetrically to the right of CDEIII extending away from CDEI and CDEII, which is consistent with previous data. The
CEN
DNA is bent ≈55° at the site of complex formation. A significant fraction of the complexes are linked in pairs, showing three to four DNA arms, with molecular volumes approximately three times the mean volumes of two-armed complexes. These multi-armed complexes indicate that CBF3 can bind two DNA molecules together
in vitro
and, thus, may be involved in holding together chromatid pairs during mitosis.
Constitutive and regulated α-secretase cleavage of Alzheimer’s amyloid precursor protein by a disintegrin metalloprotease
Authors: Lammich Sven , Kojro Elzbieta , Postina Rolf , Gilbert Sandra , Pfeiffer Roland , Jasionowski Marek , Haass Christian , Fahrenholz Falk
Published: July 2002

Amyloid β peptide (Aβ), the principal proteinaceous component of amyloid plaques in brains of Alzheimer’s disease patients, is derived by proteolytic cleavage of the amyloid precursor protein (APP). Proteolytic cleavage of APP by a putative α-secretase within the Aβ sequence precludes the formation of the amyloidogenic peptides and leads to the release of soluble APPsα into the medium. By overexpression ofa disintegrinandmetalloprotease (ADAM), classified as ADAM 10, in HEK 293 cells, basal and protein kinase C-stimulated α-secretase activity was increased severalfold. The proteolytically activated form of ADAM 10 was localized by cell surface biotinylation in the plasma membrane, but the majority of the proenzyme was found in the Golgi. These results support the view that APP is cleaved both at the cell surface and along the secretory pathway. Endogenous α-secretase activity was inhibited by a dominant negative form of ADAM 10 with a point mutation in the zinc binding site. Studies with purified ADAM 10 and Aβ fragments confirm the correct α-secretase cleavage site and demonstrate a dependence on the substrate’s conformation. Our results provide evidence that ADAM 10 has α-secretase activity and many properties expected for the proteolytic processing of APP. Increases of its expression and activity might be beneficial for the treatment of Alzheimer’s disease.
Adult brain retains the potential to generate oligodendroglial progenitors with extensive myelination capacity
Authors: Zhang Su-Chun , Ge Bin , Duncan Ian D.
Published: July 2002

Remyelination of focal areas of the central nervous system (CNS) in animals can be achieved by transplantation of glial cells, yet the source of these cells in humans to similarly treat myelin disorders is limited at present to fetal tissue. Multipotent precursor cells are present in the CNS of adult as well as embryonic and neonatal animals and can differentiate into lineage-restricted progenitors such as oligodendroglial progenitors (OPs). The OPs present in adults have a different phenotype from those seen in earlier life, and their potential role in CNS repair remains unknown. To gain insights into the potential to manipulate the myelinating capacity of these precursor and/or progenitor cells, we generated a homogenous culture of OPs from neural precursor cells isolated from adult rat subependymal tissues. Phenotypic characterization indicated that these OPs resembled neonatal rather than adult OPs and produced robust myelin after transplantation. The ability to generate such cells from the adult brain therefore opens an avenue to explore the potential of these cells for repairing myelin disorders in adulthood.
RNA polymerase switch in transcription of yeast rDNA: Role of transcription factor UAF (upstream activation factor) in silencing rDNA transcription by RNA polymerase II
Authors: Vu Loan , Siddiqi Imran , Lee Bum-Soo , Josaitis Cathleen A. , Nomura Masayasu
Published: July 2002

Transcription factor UAF (upstream activation factor) is required for a high level of transcription, but not for basal transcription, of rDNA by RNA polymerase I (Pol I) in the yeast
Saccharomyces cerevisiae. RRN9
encodes one of the UAF subunits. We have found that
rrn9
deletion mutants grow extremely slowly but give rise to faster growing variants that can grow without intact Pol I, synthesizing rRNA by using RNA polymerase II (Pol II). This change is reversible and does not involve a simple mutation. The two alternative states, one suitable for rDNA transcription by Pol I and the other favoring rDNA transcription by Pol II, are heritable not only in mitosis, but also in meiosis. Thus,
S. cerevisiae
has an inherent ability to transcribe rDNA by Pol II, but this transcription activity is silenced in normal cells, and UAF plays a key role in this silencing by stabilizing the first state.
Natural allelic variation at seed size loci in relation to other life history traits ofArabidopsis thaliana
Authors: Alonso-Blanco Carlos , Blankestijn-de Vries Hetty , Hanhart Corrie J. , Koornneef Maarten
Published: July 2002

We have analyzed twoArabidopsisstrains differing in the mean seed size and seed number they produced. The accession Cape Verde Islands (Cvi) yielded on average about 40% fewer seeds than the laboratory strain Landsbergerecta(Ler), but Cvi seeds were almost twice as heavy. Maternal and nonmaternal genetic factors were involved in the seed size variation, and interactions between both types of factors presumably occurred. The Ler/Cvi seed size difference increased through seed development from ovule maturation until seed desiccation, suggesting that multiple processes of seed development were affected. In addition, it involved changes in the final cell number and cell size of the seed coat and the embryo. Cell number variation was controlled mainly by maternal factors, whereas nonmaternal allelic variation mostly affected cell size. By using a recombinant inbred line population derived from Lerand Cvi, we mapped quantitative trait loci (QTLs) affecting 12 life history traits related to seed size, fruit size, seed number, and plant resources. Five of the seed size QTLs colocated with QTLs for other traits, suggesting that they control seed size via maternal components affecting ovule number and/or carpel development, ovule development, or reproductive resource allocation in the mother plant. The six remaining putative seed size QTLs did not show a significant effect on any other trait, suggesting that this allelic variation may be involved specifically in seed development processes.
Identification of a photoreceptor cell-specific nuclear receptor
Authors: Kobayashi Mime , Takezawa Shin-ichiro , Hara Kenji , Yu Ruth T. , Umesono Yoshihiko , Agata Kiyokazu , Taniwaki Masafumi , Yasuda Kunio , Umesono Kazuhiko
Published: July 2002

Nuclear receptors comprise a large and expanding family of transcription factors involved in diverse aspects of animal physiology and development, the functions of which can be modulated in a spatial and temporal manner by access to small lipophilic ligands and/or the specificity of their own localized expression. Here we report the identification of a human nuclear receptor that reveals a unique proximal box (CNGCSG) in the DNA-binding domain. The conservation of this feature in its nematode counterpart suggests the requirement for this type of P box in the genetic cascades mediated by nuclear receptors in a wide variety of animal species. The expression of this receptor, PNR (photoreceptor-specific nuclear receptor), appears strongly restricted in the retina, exclusively in photoreceptor cells. In human cell lines, PNR expression was observed in Y79 retinoblastoma along with other photoreceptor marker genes such as CRX. Among vertebrate receptors, PNR shares structural kinship with an orphan receptor TLX, and despite distinct differences in the DNA binding domain, PNR is able to recognize a subset of TLX target sequences
in vitro
. Analyses of the human PNR gene revealed its chromosomal position as 15q24, a site that has recently been reported as a susceptible region for retinal degeneration. These data support a role for PNR in the regulation of signalling pathways intrinsic to the photoreceptor cell function.
Biosynthesis of terpenoids: 4-Diphosphocytidyl-2C-methyl-
d
-erythritol synthase of
Arabidopsis thaliana
Authors: Rohdich Felix , Wungsintaweekul Juraithip , Eisenreich Wolfgang , Richter Gerald , Schuhr Christoph A. , Hecht Stefan , Zenk Meinhart H. , Bacher Adelbert
Published: July 2002

A hypothetical gene with similarity to the
ispD
gene of
Escherichia coli
was cloned from
Arabidopsis thaliana
cDNA. The ORF of 909 bp specifies a protein of 302 amino acid residues. The cognate chromosomal gene consists of 2,071 bp and comprises 11 introns with a size range of 78–202 bp. A fragment comprising amino acid residues 76–302 was expressed in a recombinant
E. coli
strain. The protein was purified to homogeneity and was shown to catalyze the formation of 4-diphosphocytidyl-2C-methyl-
d
-erythritol from 2C-methyl-
d
-erythritol 4-phosphate with a specific activity of 67 μmol⋅min
−1
mg
−1
. The Michaelis constants for 4-diphosphocytidyl-2C-methyl-
d
-erythritol and CTP were 500 μM and 114 μM, respectively.
Interferon regulatory factor subcellular localization is determined by a bipartite nuclear localization signal in the DNA-binding domain and interaction with cytoplasmic retention factors
Authors: Lau Joe F. , Parisien Jean-Patrick , Horvath Curt M.
Published: July 2002

The transduction of type I interferon signals to the nucleus relies on activation of a protein complex, ISGF3, involving two signal transducers and activators of transcription (STAT) proteins, STAT1 and STAT2, and the interferon (IFN) regulatory factor (IRF) protein, p48/ISGF3γ. The STAT subunits are cytoplasmically localized in unstimulated cells and rapidly translocate to the nucleus of IFN-stimulated cells, but the p48/ISGF3γ protein is found in both the nucleus and the cytoplasm, regardless of IFN stimulation. Here, we demonstrate that p48 is efficiently and constitutively targeted to the nucleus. Analysis of the subcellular distribution of green fluorescent protein-p48 fragments indicates that p48 contains a bipartite nuclear retention signal within its amino-terminal DNA-binding domain. This signal is preserved in two other IRF proteins involved in immune responses, ICSBP and IRF4. Mutations to clustered basic residues within amino acids 50–100 of p48 or IRF4 disrupt their nuclear accumulation, and DNA-binding ability is not required for nuclear targeting. This is the only example of a nuclear localization signal for any ISGF3 component and assigns a second function to the IRF DNA-binding domain. We also demonstrate that the nuclear distribution of p48 is dramatically altered by coexpression of the STAT2 protein, indicating that STAT2 forms a cytoplasmic complex with p48, overriding the intrinsic p48 nuclear targeting. Retention by STAT2 may serve to regulate the activity of free p48 and/or guarantee that cytoplasmic pools of preassociated STAT2:p48 are available for rapid activation of the IFN response. These findings suggest that analogous mechanisms may exist for regulating the distribution of other IRF proteins.
Site-directed ligand discovery
Authors: Erlanson Daniel A. , Braisted Andrew C. , Raphael Darren R. , Randal Mike , Stroud Robert M. , Gordon Eric M. , Wells James A.
Published: July 2002

We report a strategy (called “tethering”) to discover low molecular weight ligands (≈250 Da) that bind weakly to targeted sites on proteins through an intermediary disulfide tether. A native or engineered cysteine in a protein is allowed to react reversibly with a small library of disulfide-containing molecules (≈1,200 compounds) at concentrations typically used in drug screening (10 to 200 μM). The cysteine-captured ligands, which are readily identified by MS, are among the most stable complexes, even though in the absence of the covalent tether the ligands may bind very weakly. This method was applied to generate a potent inhibitor for thymidylate synthase, an essential enzyme in pyrimidine metabolism with therapeutic applications in cancer and infectious diseases. The affinity of the untethered ligand (
K
i
≈1 mM) was improved 3,000-fold by synthesis of a small set of analogs with the aid of crystallographic structures of the tethered complex. Such site-directed ligand discovery allows one to nucleate drug design from a spatially targeted lead fragment.
Highly restricted spread of HIV-1 and multiply infected cells within splenic germinal centers
Authors: Gratton Sophie , Cheynier Rémi , Dumaurier Marie-Jeanne , Oksenhendler Eric , Wain-Hobson Simon
Published: July 2002

The tremendous dynamics of HIV infection finds expression in the
tempo of sequence diversification. Genetic diversity calculations
require the clearance of a majority of infected cells, the obvious
predator being anti-HIV immune responses. Indeed, infiltration of
germinal centers (GCs) by HIV-specific CD8
+
cytotoxic T
lymphocytes has been described. A corollary to this description would
be limited diffusion of virus within lymphoid structures. HIV
efficiently infects and replicates mainly in activated CD4
+
T lymphoblasts. These cells are found within GCs after their activation
in the adjacent periarteriolar lymphoid sheath (PALS). Here GCs and
PALS have been dissected from consecutive 10-μm sections through
splenic tissue from three HIV-1-infected patients. Nested PCR
amplification of the two first hypervariable regions of the
env
gene indicated that 38–78% of sections contained
HIV-infected cells. Since there are several hundred CD4
+
T
cells per GC section, approximately 0.09–0.64% harbor proviral DNA.
Such a low frequency not only suggests that virions on the follicular
dendritic cell surfaces do not readily infect adjacent T cells but also
indicates highly restricted spread of HIV within GCs and the PALS.
Sections were heavily infiltrated by CD8
+
cells, which,
together with a large body of extant data, suggests that the majority
of infected cells are destroyed by HIV-specific cytotoxic T lymphocytes
before becoming productively infected. Finally, sequence analysis
revealed that those HIV-positive cells were multiply infected, which
helps explain widespread recombination despite a low overall frequency
of infected cells.
The mechanism by which a peptide based on complementarity-determining region-1 of a pathogenic anti-DNA auto-Ab ameliorates experimental systemic lupus erythematosus
Authors: Eilat Eran , Dayan Molly , Zinger Heidy , Mozes Edna
Published: July 2002

A peptide based on complementarity-determining region (CDR)-1 of a monoclonal murine anti-DNA Ab that bears the common idiotype, 16/6Id, was synthesized and characterized. The peptide, designated pCDR1, was found to be an immunodominant T-cell epitope in BALB/c mice. The CDR1-based peptide was shown to be capable of inhibiting thein vivopriming of BALB/c mice immunized with the peptide or with the whole anti-DNA 16/6Id+mAbs of either mouse or human origin. We show here that administration of pCDR1 (weekly, i.v., 100 μg/mouse) in aqueous solution for 5 weeks starting at the time of disease induction with the human 16/6Id prevented the development of clinical manifestations of experimental systemic lupus erythematosus (SLE). Further, 10 weekly injections of pCDR1 to BALB/c mice with an established experimental SLE down-regulated clinical manifestations of SLE (e.g., anti-DNA auto-Abs, leukopenia, proteinuria, immune complex deposits in the kidneys) in the treated mice. Prevention of SLE induction was shown to be associated mainly with a decrease in the levels of IL-2, INFγ, and the proinflammatory cytokine TNFα. On the other hand, the secretion of the immunosuppressive cytokine TGFβ was elevated. Amelioration of the clinical manifestations of an already established experimental SLE correlated with a dramatic decrease in TNFα secretion, elevated levels of TGFβ, and immunomodulation of the Th1 and Th2 type cytokines to levels close to those observed in healthy mice.
Identification and modulation of a naturally processed T cell epitope from the diabetes-associated autoantigen human glutamic acid decarboxylase 65 (hGAD65)
Authors: Nepom Gerald T. , Lippolis John D. , White Forest M. , Masewicz Susan , Marto Jarrod A. , Herman Andrew , Luckey C. John , Falk Ben , Shabanowitz Jeffrey , Hunt Donald F. , Engelhard Victor H. , Nepom Barbara S.
Published: July 2002

T cell recognition of autoantigens is critical to progressive immune-mediated destruction of islet cells, which leads to autoimmune diabetes. We identified a naturally presented autoantigen from the human islet antigen glutamic acid decarboxylase, 65-kDa isoform (GAD65), by using a combination of chromatography and mass spectrometry of peptides bound by the type I diabetes (insulin-dependent diabetes mellitus, IDDM)-associated HLA-DR4 molecule. Peptides encompassing this epitope-stimulated GAD65-specific T cells from diabetic patients and a DR4-positive individual at high risk for developing IDDM. T cell responses were antagonized by altered peptide ligands containing single amino acid modifications. This direct identification and manipulation of GAD65 epitope recognition provides an approach toward dissection of the complex CD4+T cell response in IDDM.
Hypoxia-induced gene expression profiling in the euryoxic fish
Gillichthys mirabilis
Authors: Gracey Andrew Y. , Troll Joshua V. , Somero George N.
Published: July 2002

Hypoxia is important in both biomedical and environmental contexts
and necessitates rapid adaptive changes in metabolic organization.
Mammals, as air breathers, have a limited capacity to withstand
sustained exposure to hypoxia. By contrast, some aquatic animals, such
as certain fishes, are routinely exposed and resistant to severe
environmental hypoxia. Understanding the changes in gene expression in
fishes exposed to hypoxic stress could reveal novel mechanisms of
tolerance that may shed new light on hypoxia and ischemia in higher
vertebrates. Using cDNA microarrays, we have studied gene expression in
a hypoxia-tolerant burrow-dwelling goby fish,
Gillichthys
mirabilis.
We show that a coherent picture of a complex
transcriptional response can be generated for a nonmodel organism for
which sequence data were unavailable. We demonstrate that:
(
i
) although certain shifts in gene expression mirror
changes in mammals, novel genes are differentially expressed in fish;
and (
ii
) tissue-specific patterns of expression reflect
the different metabolic roles of tissues during hypoxia.
Modernization and religious intermarriage in the Republic of Ireland
Authors: O'Leary Richard
Published: July 2002

There are strong theoretical reasons for hypothesizing that those sections of the population who are most exposed to modernization processes are more likely to marry outside their own religious group. We examine this for Catholic‐Protestant intermarriage in the Republic of Ireland. We use a multivariate analysis on survey data to test the hypotheses that urban dwellers, persons of non‐farming parents, persons with higher levels of education and the young are more likely to be religiously intermarried. While the odds of being in an intermarriage are greater for urban dwellers, this is in large part due to their being non‐farmers. The farming effect is not necessarily related to religiosity. The odds of being in an intermarriage do not increase significantly for persons with third level education and this can be explained in terms of the marriage market for minority groups. It is shown that the historical context and the minority position of religious group should be taken into account in explanations which relate modernization to the pattern of religious intermarriage.
Cross‐cultural Differences in Self‐reported Decision‐making Style and Confidence
Authors: Mann Leon , Radford Mark , Burnett Paul , Ford Steve , Bond Michael , Leung Kwok , Nakamura Hiyoshi , Vaughan Graham , Yang Kuo‐Shu
Published: July 2002

The Melbourne Decision Making Questionnaire (Mann, Burnett, Radford, & Ford, 1997) measures self‐reported decision‐making coping patterns. The questionnaire was administered to samples of University students in the US (N = 475), Australia (N = 262), New Zealand (N = 260), Japan (N = 359), Hong Kong (N = 281), and Taiwan (N = 414). As predicted, students from the three Western, individualistic cultures (US, Australia, and New Zealand) were more confident of their decision‐making ability than students from the three East Asian, group‐oriented cultures (Japan, Hong Kong, Taiwan). No cross‐cultural differences were found in scores on decision vigilance (a careful decision‐making style). However, compared with Western students, the Asian students tended to score higher on buck‐passing and procrastination (avoidant styles of decision making) as well as hypervigilance (a panicky style of decision making). Japanese students scored lowest on decision self‐esteem and highest on procrastination and hypervigilance. It was argued that the conflict model and its attendant coping patterns is relevant for describing and comparing decision making in both Western and Asian cultures.
An Analysis of Perceptions Concerning the Environmental Quality of Housing in Geneva
Authors: Bender Andre , Din Allan , Favarger Philippe , Hoesli Martin , Laakso Janne
Published: July 2002

The aim of this paper is to gain a better understanding of the characteristics related to the environment of single-family houses in the greater Geneva area. An Analytical Hierarchy Process (AHP) methodology is applied to the data collected by means of a questionnaire which was sent to 850 owners of houses in Geneva. The pairwise comparisons are done with eight criteria. For the 28 comparisons, the standard levels of preferences are used, but with a multiplicative scale rather than the standard linear scale. The results show that distance to a green area and quietness of the area are the two most important factors. Another finding is that the proximity of shopping centres and schools is not as important as in other countries.
“The Czech Nation must be Catholic!” An Alternative version of Czech Nationalism during the First Republic
Authors: Paces Cynthia J.
Published: July 2002

Jaroslav Durych, a popular Czech Catholic poet and essayist, began his weekly column in Lidové listy's (People's News) 10 May 1923 issue with the following proclamation: “The Czech Nation must be Catholic!” What did Durych mean by this puzzling statement? The majority of Czechs in the new Czechoslovak state considered themselves at least nominally Catholic. Yet Durych's article did not address the confessional status of Czechoslovakia's population, nor did it address religious differences between Czechs and Slovaks. Instead, Durych concerned himself with the representation of the Czech nation in popular mythology and official symbolism. He demanded that the Czech national symbols reflect the country's majority religion and not the Protestant experiment of the late Middle Ages.
French parents’ cultural models and childrearing beliefs
Authors: Suizzo Marie-Anne
Published: July 2002

This study explored, identified, and described “cultural models” of parenting shared by Parisian parents of infants and young children from birth to age 3 years. A questionnaire was constructed in the field to assess levels of importance attached to 50 practices associated with the daily care of infants and young children. Data were collected from a sample of 455 Parisian mothers and fathers and principal components analysis was used to identify three reliable components of parenting beliefs that constituted cultural models: “Awakening and exposing child to diverse stimuli”, “Ensuring proper presentation of child”, and “Responding to and bonding with child”. Four sociodemographic variables predicted variation in mean importance scores associated with each of the three cultural models: parent’s age, gender, education level, and religiosity, although the magnitudes of these associations were small. The discussion highlights the strengths and limitations of this method and offers suggestions for further research.
Psychosocial influences on possible selves: A comparison of three cohorts of older adults
Authors: Frazier Leslie D. , Johnson Paulette M. , Gonzalez Gema K. , Kafka Carissa L.
Published: July 2002

This study examined the psychosocial factors that influence the possible selves of older adults in their 60s, 70s, and 80s or older. Specifically, the factors that influence when and why health becomes salient in later life were examined. Findings showed that health was the most important domain of self for the oldest group. Although age was the strongest predictor of health-related selves, older males with better health and few physical limitations, were most likely to report health-related selves. Leisure, the most salient domain for the youngest group, was predicted by age, independent of health. The findings from this study shed light on how sense of self is influenced by the changing developmental context of the later years, the importance of exploring alternative conceptions of ageing, and help to identify possible points of intervention to help older adults age more successfully.
Explicitness and Conservation: Social Class Differences
Authors: Roazzi Antonio , Bryant Peter
Published: July 2002

The performance of 5-, 6-, 7-, and 8-year-old children in liquid conservation tasks was studied in four conditions. In the first two conditions (Standard and Incidental) the initial comparison in the task was made perceptually. In the other two conditions (Quantity and Money) the child was not allowed to make a direct perceptual comparison and the initial comparison was made by measurement. The children did much better when they measured the quantities than when they simply made perceptual comparisons, and this effect was stronger with working class children than with middle class children. Contrary to previous reports, there was no difference between the Standard and the Incidental conditions. We conclude that children in general, and working class children in particular, are helped when the nature of the task is made more explicit.
Determinants of Behavioural Problems in Romanian Children Adopted in Ontario
Authors: Marcovitch Sharon , Goldberg Susan , Gold Amanda , Washington Jane , Wasson Christine , Krekewich Karla , Handley-Derry Mark
Published: July 2002

Assessments of developmental status, attachment, and behaviour problems were conducted for 56 Romanian orphans adopted in Ontario. The group as a whole was functioning in the normal range and was considered well adjusted, but children who had experienced less than six months of institutional care had better outcomes than the rest on developmental measures. The adoptees showed an unusual distribution of attachment patterns: Secure attachment was less frequent than normally expected and avoidant attachment was not observed. Unexpectedly, neither age at adoption nor length of institutionalisation was related to attachment outcomes and it was suggested that the present preschool attachment system does not adequately capture attachment phenomena in this sample. Children who had more institution experience, those who were developmentally less competent, and those who were insecurely attached had more parent-reported behaviour problems.
Retrospective Survey of Parental Marital Relations and Child Reproductive Development
Authors: Kim Kenneth , Smith Peter K.
Published: July 2002

Belsky, Steinberg, and Draper (1991) predicted that early childhood stress or conflict in the family environment would be associated with childhood behavioural symptoms, early puberty, and early, less discriminate reproductive behaviour. A cross-sectional self-report survey of childhood family life and adolescent development was carried out with 357 university students aged 18 to 24 from Toronto, Canada. In women, earlier menarche was associated with more parental marital conflict in early childhood (birth to age 7), more parental marital unhappiness throughout childhood (birth to age 11), more independence from mother or father in late childhood (age 8 to 11), less anxiousness or internalising symptoms (anxiousness/depression) in late childhood (age 8 to 11), earlier age at dating men, and more boyfriends. In men, earlier spermarche was associated with father absence (birth to before spermarche), more stress in quality of family life, parental marital unhappiness, and parental marital conflict in early childhood (birth to age 7), more independence from mother or father in late childhood (age 8 to 11), earlier age at dating women, more girlfriends, and earlier age at sexual intercourse. These ndings are generally consistent with the Belsky et al. (1991) view that childhood psychosocial stresses affect puberty and reproductive life history, though they do not preclude alternative accounts.
Evidence for the Existence of Insect Defensin‐Like Peptide in Scorpion Venom
Authors: Zhu Shunyi , Li Wenxin , Jiang Dahe , Zeng Xianchun
Published: July 2002

Insect defensin refers to a group of antibacterial peptides derived from a variety of insect species as well as from scorpion and possessing a three‐dimensional structure highly similar to that of scorpion toxins. A full‐length cDNA encoding an insect defensin‐like peptide was isolated from the venom gland cDNA library of the Chinese scorpion
Structural Requirements for Basolateral Sorting of the Human Transferrin Receptor in the Biosynthetic and Endocytic Pathways of Madin-Darby Canine Kidney Cells
Authors: Odorizzi Greg , Trowbridge Ian S.
Published: July 2002

In polarized Madin-Darby canine kidney (MDCK) cells, the transferrin receptor (TR) is selectively delivered to the basolateral surface, where it internalizes transferrin via clathrin-coated pits and recycles back to the basolateral border. Mutant tailless receptors are sorted randomly in both the biosynthetic and endocytic pathways, indicating that the basolateral sorting of TR is dependent upon a signal located within the 61–amino acid cytoplasmic domain. To identify the basolateral sorting signal of TR, we have analyzed a series of mutant human TR expressed in MDCK cells. We find that residues 19–41 are sufficient for basolateral sorting from both the biosynthetic and endocytic pathways and that this is the only region of the TR cytoplasmic tail containing basolateral sorting information. The basolateral sorting signal is distinct from the YTRF internalization signal contained within this region and is not tyrosine based. Detailed functional analyses of the mutant TR indicate that residues 29–35 are the most important for basolateral sorting from the biosynthetic pathway. The structural requirements for basolateral sorting of internalized receptors from the endocytic pathway are not identical. The most striking difference is that alteration of G31DNS34 to YTRF impairs basolateral sorting of newly synthesized receptors from the biosynthetic pathway but not internalized receptors from the endocytic pathway. Also, mutations have been identified that selectively impair basolateral sorting of internalized TRs from the endocytic pathway without affecting basolateral sorting of newly synthesized receptors. These results imply that there are subtle differences in the recognition of the TR basolateral sorting signal by separate sorting machinery located within the biosynthetic and endocytic pathways.
Plasma Membrane Translocation of Fluorescent-labeled Phosphatidylethanolamine Is Controlled by Transcription Regulators, PDR1 and PDR3
Authors: Kean Leslie S. , Grant Althea M. , Angeletti Cesar , Mahé Yannick , Kuchler Karl , Fuller Robert S. , Nichols J. Wylie
Published: July 2002

The transcription regulators, PDR1 and PDR3, have been shown to activate the transcription of numerous genes involved in a wide range of functions, including resistance to physical and chemical stress, membrane transport, and organelle function in Saccharomyces cerevisiae. We report here that PDR1 and PDR3 also regulate the transcription of one or more undetermined genes that translocate endogenous and fluorescent-labeled (M-C6-NBD-PE) phosphatidylethanolamine across the plasma membrane. A combination of fluorescence microscopy, fluorometry, and quantitative analysis demonstrated that M-C6-NBD-PE can be translocated both inward and outward across the plasma membrane of yeast cells. Mutants, defective in the accumulation of M-C6-NBD-PE, were isolated by selectively photokilling normal cells that accumulated the fluorescent phospholipid. This led to the isolation of numerous trafficking in phosphatidylethanolamine (tpe) mutants that were defective in intracellular accumulation of M-C6-NBD-PE. Complementation cloning and linkage analysis led to the identification of the dominant mutation TPE1-1 as a new allele of PDR1 and the semidominant mutation tpe2-1 as a new allele of PDR3. The amount of endogenous phosphatidylethanolamine exposed to the outer leaflet of the plasma membrane was measured by covalent labeling with the impermeant amino reagent, trinitrobenzenesulfonic acid. The amount of outer leaflet phosphatidylethanolamine in both mutant strains increased four- to fivefold relative to the parent Tpe+ strain, indicating that the net inward flux of endogenous phosphatidylethanolamine as well as M-C6-NBD-PE was decreased. Targeted deletions of PDR1 in the new allele, PDR1-11, and PDR3 in the new allele, pdr3-11, resulted in normal M-C6-NBD-PE accumulation, confirming that PDR1-11 and pdr3-11 were gain-of-function mutations in PDR1 and PDR3, respectively. Both mutant alleles resulted in resistance to the drugs cycloheximide, oligomycin, and 4-nitroquinoline N-oxide (4-NQO). However, a previously identified drug-resistant allele, pdr3-2, accumulated normal amounts of M-C6-NBD-PE, indicating allele specificity for the loss of M-C6-NBD-PE accumulation. These data demonstrated that PDR1 and PDR3 regulate the net rate of M-C6-NBD-PE translocation (flip-flop) and the steady-state distribution of endogenous phosphatidylethanolamine across the plasma membrane.
Light Chain– dependent Regulation of Kinesin's Interaction with Microtubules
Authors: Verhey Kristen J. , Lizotte Donna L. , Abramson Tatiana , Barenboim Linda , Schnapp Bruce J. , Rapoport Tom A.
Published: July 2002

We have investigated the mechanism by which conventional kinesin is prevented from binding to microtubules (MTs) when not transporting cargo. Kinesin heavy chain (HC) was expressed in COS cells either alone or with kinesin light chain (LC). Immunofluorescence microscopy and MT cosedimentation experiments demonstrate that the binding of HC to MTs is inhibited by coexpression of LC. Association between the chains involves the LC NH2-terminal domain, including the heptad repeats, and requires a region of HC that includes the conserved region of the stalk domain and the NH2 terminus of the tail domain. Inhibition of MT binding requires in addition the COOH-terminal 64 amino acids of HC. Interaction between the tail and the motor domains of HC is supported by sedimentation experiments that indicate that kinesin is in a folded conformation. A pH shift from 7.2 to 6.8 releases inhibition of kinesin without changing its sedimentation behavior. Endogenous kinesin in COS cells also shows pH-sensitive inhibition of MT binding. Taken together, our results provide evidence that a function of LC is to keep kinesin in an inactive ground state by inducing an interaction between the tail and motor domains of HC; activation for cargo transport may be triggered by a small conformational change that releases the inhibition of the motor domain for MT binding.
Neurotransmitter Secretion along Growing Nerve Processes: Comparison with Synaptic Vesicle Exocytosis
Authors: Zakharenko Stanislav , Chang Sunghoe , O'Donoghue Michael , Popov Sergey V.
Published: July 2002

In mature neurons, synaptic vesicles continuously recycle within the presynaptic nerve terminal. In developing axons which are free of contact with a postsynaptic target, constitutive membrane recycling is not localized to the nerve terminal; instead, plasma membrane components undergo cycles of exoendocytosis throughout the whole axonal surface (Matteoli et al., 1992; Kraszewski et al., 1995). Moreover, in growing Xenopus spinal cord neurons in culture, acetylcholine (ACh) is spontaneously secreted in the quantal fashion along the axonal shaft (Evers et al., 1989; Antonov et al., 1998). Here we demonstrate that in Xenopus neurons ACh secretion is mediated by vesicles which recycle locally within the axon. Similar to neurotransmitter release at the presynaptic nerve terminal, ACh secretion along the axon could be elicited by the action potential or by hypertonic solutions. We found that the parameters of neurotransmitter secretion at the nerve terminal and at the middle axon were strikingly similar. These results lead us to conclude that, as in the case of the presynaptic nerve terminal, synaptic vesicles involved in neurotransmitter release along the axon contain a complement of proteins for vesicle docking and Ca2+-dependent fusion. Taken together, our results support the idea that, in developing axons, the rudimentary machinery for quantal neurotransmitter secretion is distributed throughout the whole axonal surface. Maturation of this machinery in the process of synaptic development would improve the fidelity of synaptic transmission during high-frequency stimulation of the presynaptic cell.
Phosphorylation Regulates In Vivo Interaction and Molecular Targeting of Serine/Arginine-rich Pre-mRNA Splicing Factors
Authors: Yeakley Joanne M. , Tronchère Hélène , Olesen James , Dyck Jacqueline A. , Wang Huan-You , Fu Xiang-Dong
Published: July 2002

The SR superfamily of splicing factors and regulators is characterized by arginine/serine (RS)-rich domains, which are extensively modified by phosphorylation in cells. In vitro binding studies revealed that RS domain–mediated protein interactions can be differentially affected by phosphorylation. Taking advantage of the single nonessential SR protein–specific kinase Sky1p in Saccharomyces cerevisiae, we investigated RS domain interactions in vivo using the two-hybrid assay. Strikingly, all RS domain–mediated interactions were abolished by SKY1 deletion and were rescuable by yeast or mammalian SR protein–specific kinases, indicating that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro. To understand this dramatic effect, we examined the localization of SR proteins and found that SC35 was shifted to the cytoplasm in sky1Δ yeast, although this phenomenon was not obvious with ASF/SF2, indicating that nuclear import of SR proteins may be differentially regulated by phosphorylation. Using a transcriptional repression assay, we further showed that most LexA-SR fusion proteins depend on Sky1p to efficiently recognize the LexA binding site in a reporter, suggesting that molecular targeting of RS domain–containing proteins within the nucleus was also affected. Together, these results reveal multiple phosphorylation-dependent steps for SR proteins to interact with one another efficiently and specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.
Shedding of Syndecan-1 and -4 Ectodomains Is Regulated by Multiple Signaling Pathways and Mediated by a Timp-3–Sensitive Metalloproteinase
Authors: Fitzgerald Marilyn L. , Wang Zihua , Park Pyong Woo , Murphy Gillian , Bernfield Merton
Published: July 2002

The syndecan family of four transmembrane heparan sulfate proteoglycans binds a variety of soluble and insoluble extracellular effectors. Syndecan extracellular domains (ectodomains) can be shed intact by proteolytic cleavage of their core proteins, yielding soluble proteoglycans that retain the binding properties of their cell surface precursors. Shedding is accelerated by PMA activation of protein kinase C, and by ligand activation of the thrombin (G-protein–coupled) and EGF (protein tyrosine kinase) receptors (Subramanian, S.V., M.L. Fitzgerald, and M. Bernfield. 1997. J. Biol. Chem. 272:14713–14720). Syndecan-1 and -4 ectodomains are found in acute dermal wound fluids, where they regulate growth factor activity (Kato, M., H. Wang, V. Kainulainen, M.L. Fitzgerald, S. Ledbetter, D.M. Ornitz, and M. Bernfield. 1998. Nat. Med. 4:691–697) and proteolytic balance (Kainulainen, V., H. Wang, C. Schick, and M. Bernfield. 1998. J. Biol. Chem. 273:11563–11569). However, little is known about how syndecan ectodomain shedding is regulated.

To elucidate the mechanisms that regulate syndecan shedding, we analyzed several features of the process that sheds the syndecan-1 and -4 ectodomains. We find that shedding accelerated by various physiologic agents involves activation of distinct intracellular signaling pathways; and the proteolytic activity responsible for cleavage of syndecan core proteins, which is associated with the cell surface, can act on unstimulated adjacent cells, and is specifically inhibited by TIMP-3, a matrix-associated metalloproteinase inhibitor. In addition, we find that the syndecan-1 core protein is cleaved on the cell surface at a juxtamembrane site; and the proteolytic activity responsible for accelerated shedding differs from that involved in constitutive shedding of the syndecan ectodomains. These results demonstrate the existence of highly regulated mechanisms that can rapidly convert syndecans from cell surface receptors or coreceptors to soluble heparan sulfate proteoglycan effectors. Because the shed ectodomains are found and function in vivo, regulation of syndecan ectodomain shedding by physiological mediators indicates that shedding is a response to specific developmental and pathophysiological cues.
Functional Differences in Yeast Protein Disulfide Isomerases
Authors: Nørgaard Per , Westphal Vibeke , Tachibana Christine , Alsøe Lene , Holst Bjørn , Winther Jakob R.
Published: July 2002

PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several cases, we found that the ability of the PDI1 homologues to restore viability to a pdi1-deleted strain when overexpressed was dependent on the presence of low endogenous levels of one or more of the other homologues. This shows that the homologues are not functionally interchangeable. In fact, Mpd1p was the only homologue capable of carrying out all the essential functions of Pdi1p. Furthermore, the presence of endogenous homologues with a CXXC motif in the thioredoxin-like domain is required for suppression of a pdi1 deletion by EUG1 (which contains two CXXS active site motifs). This underlines the essentiality of protein disulfide isomerase-catalyzed oxidation. Most mutant combinations show defects in carboxypeptidase Y folding as well as in glycan modification. There are, however, no significant effects on ER-associated protein degradation in the various protein disulfide isomerase-deleted strains.
Distribution and Function of Ap-1 Clathrin Adaptor Complexes in Polarized Epithelial Cells
Authors: Fölsch Heike , Pypaert Marc , Schu Peter , Mellman Ira
Published: July 2002

Expression of the epithelial cell–specific heterotetrameric adaptor complex AP-1B is required for the polarized distribution of many membrane proteins to the basolateral surface of LLC-PK1 kidney cells. AP-1B is distinguished from the ubiquitously expressed AP-1A by exchange of its single 50-kD μ subunit, μ1A, being replaced by the closely related μ1B. Here we show that this substitution is sufficient to couple basolateral plasma membrane proteins, such as a low-density lipoprotein receptor (LDLR), to the AP-1B complex and to clathrin. The interaction between LDLR and AP-1B is likely to occur in the trans-Golgi network (TGN), as was suggested by the localization of functional, epitope-tagged μ1 by immunofluorescence and immunoelectron microscopy. Tagged AP-1A and AP-1B complexes were found in the perinuclear region close to the Golgi complex and recycling endosomes, often in clathrin-coated buds and vesicles. Yet, AP-1A and AP-1B localized to different subdomains of the TGN, with only AP-1A colocalizing with furin, a membrane protein that uses AP-1 to recycle between the TGN and endosomes. We conclude that AP-1B functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes.