bluuebunny/crossref_metadata_2025 huggingface dataset:
Exceptionally fast self-cleavage by a
Neurospora
Varkud satellite ribozyme
Authors: Zamel Ricardo , Poon Alan , Jaikaran Dominic , Andersen Angela , Olive Joan , De Abreu Diane , Collins Richard A.
Published: February 2004

Most of the small ribozymes, including those that have been investigated as potential therapeutic agents, appear to be rather poor catalysts. These RNAs use an internal phosphoester transfer mechanism to catalyze site-specific RNA cleavage with apparent cleavage rate constants typically <2 min
-1
. We have identified variants of one of these, the
Neurospora
Varkud satellite ribozyme, that self-cleaves with experimentally measured apparent rate constants of up to 10 s
-1
(600 min
-1
), ≈2 orders of magnitude faster than any previously characterized self-cleaving RNA. We describe structural features of the cleavage site loop and an adjacent helix that affect the apparent rate constants for cleavage and ligation and the equilibrium between them. These data show that the phosphoester transfer ribozymes can catalyze reactions with rate constants much larger than previously appreciated and in the range of those of protein enzymes that perform similar reactions.
Glucose sensing and signaling in
Saccharomyces cerevisiae
through the Rgt2 glucose sensor and casein kinase I
Authors: Moriya Hisao , Johnston Mark
Published: February 2004

The yeast
Saccharomyces cerevisiae
senses glucose through two transmembrane glucose sensors, Snf3 and Rgt2. Extracellular glucose causes these sensors to generate an intracellular signal that induces expression of
HXT
genes encoding glucose transporters by inhibiting the function of Rgt1, a transcriptional repressor of
HXT
genes. We present the following evidence that suggests that the glucose sensors are coupled to the membrane-associated protein kinase casein kinase I (Yck1). (
i
) Overexpression of Yck1 leads to constitutive
HXT1
expression; (
ii
) Yck1 (or its paralogue Yck2) is required for glucose induction of
HXT1
expression; (
iii
) Yck1 interacts with the Rgt2 glucose sensor; and (
iv
) attaching the C-terminal cytoplasmic tail of Rgt2 to Yck1 results in a constitutive glucose signal. The likely targets of Yck1 in this signal transduction pathway are Mth1 and Std1, which bind to and regulate function of the Rgt1 transcription factor and bind to the C-terminal cytoplasmic domain of glucose sensors. Potential casein kinase I phosphorylation sites in Mth1 and Std1 are required for normal glucose regulation of
HXT1
expression, and Yck1 catalyzes phosphorylation of Mth1 and Std1
in vitro
. These results support a model of glucose signaling in which glucose binding to the glucose sensors causes them to activate Yck1 in the cell membrane, which then phosphorylates Mth1 and Std1 bound to the cytoplasmic face of the glucose sensors, triggering their degradation and leading to the derepression of
HXT
gene expression. Our results add nutrient sensing to the growing list of processes in which casein kinase I is involved.
Automated protein crystal structure determination using
elves
Authors: Holton James , Alber Tom
Published: February 2004

Efficient determination of protein crystal structures requires automated x-ray data analysis. Here, we describe the expert system
elves
and its use to determine automatically the structure of a 12-kDa protein. Multiwavelength anomalous diffraction analysis of a selenomethionyl derivative was used to image the Asn-16-Ala variant of the GCN4 leucine zipper. In contrast to the parallel, dimeric coiled coil formed by the WT sequence, the mutant unexpectedly formed an antiparallel trimer. This structural switch reveals how avoidance of core cavities at a single site can select the native fold of a protein. All structure calculations, including indexing, data processing, locating heavy atoms, phasing by multiwavelength anomalous diffraction, model building, and refinement, were completed without human intervention. The results demonstrate the feasibility of automated methods for determining high-resolution, x-ray crystal structures of proteins.
Control of late off-center cone bipolar cell differentiation and visual signaling by the homeobox gene
Vsx1
Authors: Chow Robert L. , Volgyi Bela , Szilard Rachel K. , Ng David , McKerlie Colin , Bloomfield Stewart A. , Birch David G. , McInnes Roderick R.
Published: February 2004

Retinal bipolar cells are interneurons that transmit visual signals from photoreceptors to ganglion cells. Although the visual pathways mediated by bipolar cells have been well characterized, the genes that regulate their development and function are largely unknown. To determine the role in bipolar cell development of the homeobox gene
Vsx1
, whose retinal expression is restricted to a major subset of differentiating and mature cone bipolar (CB) cells, we targeted the gene in mice. Bipolar cell fate was not altered in the absence of
Vsx1
function, because the pan-bipolar markers Chx10 and Ret-B1 continued to be expressed in inner nuclear layer neurons labeled by the
Vsx1
-targeting reporter gene, τ
LacZ
. The specification, number, and gross morphology of the subset of on-center and off-center (OFF)-CB cells defined by τ
LacZ
expression from the
Vsx1
locus were also normal in
Vsx1

τ
LacZ

/
Vsx1

τ
LacZ

mice. However, the terminal differentiation of OFF-CB cells in the retina of
Vsx1

τ
LacZ

/
Vsx1

τ
LacZ

mice was incomplete, as demonstrated by a substantial reduction in the expression of at least four markers (recoverin, NK3R, Neto1, and CaB5) for these interneurons. These molecular abnormalities were associated with defects in retinal function and documented by electroretinography and
in vitro
ganglion cell recordings specific to cone visual signaling. In particular, there was a general reduction in the light-mediated activity of OFF, but not on-center, ganglion cells. Thus,
Vsx1
is required for the late differentiation and function of OFF-CB cells and is associated with a heritable OFF visual pathway-specific retinal defect.
Long-term effects of culture of preimplantation mouse embryos on behavior
Authors: Ecker David J. , Stein Paula , Xu Zhe , Williams Carmen J. , Kopf Gregory S. , Bilker Warren B. , Abel Ted , Schultz Richard M.
Published: February 2004

Many procedures used in assisted reproductive technologies (ART) to treat human infertility entail culture of preimplantation embryos. Moreover, there is an increasing trend to culture embryos for longer periods of time before uterine transfer to identify the “best” embryos for transfer and to minimize multiple pregnancies. Embryo culture, however, can perturb embryo metabolism and gene expression, and the long-term consequences of culture are unknown. We have explored the behavioral consequences of embryo culture by using a 129S6/SvEvTac/C57BL/6J F1mouse model and find that adults derived from cultured embryos exhibit specific behavioral alterations in the elevated zero maze and Morris water maze tasks.
Reorganization of the auditory cortex specialized for echo-delay processing in the mustached bat
Authors: Xiao Zhongju , Suga Nobuo
Published: February 2004

Focal excess sensory stimulation evokes reorganization of a sensory system. It is usually an expansion of the neural representation of that stimulus resulting from the shifts of the tuning curves (receptive fields) of neurons toward those of the stimulated neurons. The auditory cortex of the mustached bat has an area that is highly specialized for the processing of target-distance information carried by echo delays. In this area, however, reorganization is due to shifts of the delay-tuning curves of neurons away from those of the stimulated cortical neurons. Elimination of inhibition in the target-distance processing area in the auditory cortex by a drug reverses the direction of the shifts in neural tuning curves. Therefore, such unique reorganization in the time domain is due to strong lateral inhibition in the highly specialized area of the auditory cortex.
Herpes simplex virus infections are arrested in Oct-1-deficient cells
Authors: Nogueira Mauricio L. , Wang Victoria E. H. , Tantin Dean , Sharp Phillip A. , Kristie Thomas M.
Published: February 2004

Expression of the herpes simplex virus (HSV) immediate early (IE) genes is regulated by a multiprotein complex that is assembled on the TAATGARAT enhancer core element. The complex contains the cellular POU domain protein Oct-1, the viral transactivator VP16, and the cellular cofactor host cell factor 1. The current model suggests that the assembly depends on recognition of the core element by Oct-1. Here, HSV infection of Oct-1-deficient mouse embryonic fibroblast cells demonstrates that Oct-1 is critical for IE gene expression at low multiplicities of infection (moi). However, the protein is not essential for IE gene expression at high moi, indicating that VP16-mediated transcriptional induction through other IE regulatory elements is also important. This induction depends, at least in part, on the GA-binding protein binding elements that are present in each IE enhancer domain. Surprisingly, whereas the viral IE genes are expressed after high moi infection of Oct-1-deficient cells, the assembly of viral replication factories is severely impaired, revealing a second critical role for Oct-1 in HSV replication. The results have implications for both the HSV lytic and latency-reactivation cycles.
Genomic identification of direct target genes of LEAFY
Authors: William Dilusha A. , Su Yanhui , Smith Michael R. , Lu Meina , Baldwin Don A. , Wagner Doris
Published: February 2004

The switch from vegetative to reproductive development in plants necessitates a switch in the developmental program of the descendents of the stem cells in the shoot apical meristem. Genetic and molecular investigations have demonstrated that the plant-specific transcription factor and meristem identity regulator LEAFY (LFY) controls this developmental transition by inducing expression of a second transcription factor, APETALA1, and by regulating the expression of additional, as yet unknown, genes. Here we show that the additional LFY targets include the APETALA1-related factor, CAULI-FLOWER, as well as three transcription factors and two putative signal transduction pathway components. These genes are up-regulated by LFY even when protein synthesis is inhibited and, hence, appear to be direct targets of LFY. Supporting this conclusion, cis-regulatory regions upstream of these genes are bound by LFY
in vivo
. The newly identified LFY targets likely initiate the transcriptional changes that are required for the switch from vegetative to reproductive development in
Arabidopsis
.
Human endogenous retrovirus K solo-LTR formation and insertional polymorphisms: Implications for human and viral evolution
Authors: Hughes Jennifer F. , Coffin John M.
Published: February 2004

Human endogenous retroviruses (HERVs) are a potential source of genetic diversity in the human genome. Although many of these elements have been inactivated over time by the accumulation of deleterious mutations or internal recombination leading to solo-LTR formation, several members of the HERV-K family have been identified that remain nearly intact and probably represent recent integration events. To determine whether HERV-K elements have caused recent changes in the human genome, we have undertaken a study of the level of HERV-K polymorphism that exists in the human population. By using a high-resolution unblotting technique, we analyzed 13 human-specific HERV-K elements in 18 individuals. We found that solo LTRs have formed at five of these loci. These results enable the estimation of HERV solo-LTR formation in the human genome and indicate that these events occur much more frequently than described in inbred mice. Detailed sequence analysis of one provirus shows that solo-LTR formation occurred at least three separate times in recent history. An unoccupied preintegration site also was present at this locus in two individuals, indicating that although the age of this provirus is estimated to be ≈1.2 million years, it has not yet become fixed in the human population.
Autoimmune glomerulonephritis with spontaneous formation of splenic germinal centers in mice lacking theestrogen receptor alphagene
Authors: Shim Gil-Jin , Kis Loránd Levente , Warner Margaret , Gustafsson Jan-Åke
Published: February 2004

In mice, ovariectomy accelerates the progression of the end-stage renal disease glomerulosclerosis. In women, the incidence of this disease increases after menopause, and estrogen alters its progression. Polymorphisms in the human estrogen receptor α (ERα) gene have been suggested to constitute a genetic predisposition for lupus nephritis. Here we show that by 1 year of age, mice lacking ERα (ERα-/-) but not those lacking ERβ (ERβ-/-) exhibit immune complex-type glomerulonephritis, proteinuria, and destruction of tubular cells with severe infiltration of B lymphocytes in the kidney and the presence of anti-DNA antibodies in serum. No gender difference occurred in the incidence or severity of these symptoms. However, in female but not in male ERα-/-mice there were elevated serum levels of IgG3. Other prominent features of these mice were (i) spontaneous formation of germinal centers in the spleen in the absence of antigen challenge and (ii) infiltration of plasma cells in the kidney and plasmacytosis in the spleen. Immunohistochemistry indicated a selective expression of ERα protein in the germinal centers but not in the follicular mantle zone of murine spleens and human tonsils. Our results indicate that ERα has indispensable functions in the kidney and in germinal centers, and that defective ERα signaling results in glomerulonephritis.
Major events in the genome evolution of vertebrates: Paranome age and size differ considerably between ray-finned fishes and land vertebrates
Authors: Vandepoele Klaas , De Vos Wouter , Taylor John S. , Meyer Axel , Van de Peer Yves
Published: February 2004

It has been suggested that fish have more genes than humans. Whether most of these additional genes originated through a complete (fish-specific) genome duplication or through many lineage-specific tandem gene or smaller block duplications and family expansions continues to be debated. We analyzed the complete genome of the pufferfish
Takifugu rubripes
(Fugu) and compared it with the paranome of humans. We show that most paralogous genes of Fugu are the result of three complete genome duplications. Both relative and absolute dating of the complete predicted set of protein-coding genes suggest that initial genome duplications, estimated to have occurred at least 600 million years ago, shaped the genome of all vertebrates. In addition, analysis of >150 block duplications in the Fugu genome clearly supports a fish-specific genome duplication (≈320 million years ago) that coincided with the vast radiation of most modern ray-finned fishes. Unlike the human genome, Fugu contains very few recently duplicated genes; hence, many human genes are much younger than fish genes. This lack of recent gene duplication, or, alternatively, the accelerated rate of gene loss, is possibly one reason for the drastic reduction of the genome size of Fugu observed during the past 100 million years or so, subsequent to the additional genome duplication that ray-finned fishes but not land vertebrates experienced.
Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in
Arabidopsis
Authors: Kim Sung-Jin , Kim Mi-Ran , Bedgar Diana L. , Moinuddin Syed G. A. , Cardenas Claudia L. , Davin Laurence B. , Kang ChulHee , Lewis Norman G.
Published: February 2004

Of 17 genes annotated in the
Arabidopsis
genome database as cinnamyl alcohol dehydrogenase (CAD) homologues, an
in silico
analysis revealed that 8 genes were misannotated. Of the remaining nine, six were catalytically competent for NADPH-dependent reduction of
p
-coumaryl, caffeyl, coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes, whereas three displayed very low activity and only at very high substrate concentrations. Of the nine putative CADs, two (AtCAD5 and AtCAD4) had the highest activity and homology (≈83% similarity) relative to bona fide CADs from other species. AtCAD5 used all five substrates effectively, whereas AtCAD4 (of lower overall catalytic capacity) poorly used sinapyl aldehyde; the corresponding 270-fold decrease in
k
enz
resulted from higher
K
m
and lower
k
cat
values, respectively. No CAD homologue displayed a specific requirement for sinapyl aldehyde, which was in direct contrast with unfounded claims for a so-called sinapyl alcohol dehydrogenase in angiosperms. AtCAD2, 3, as well as AtCAD7 and 8 (highest homology to sinapyl alcohol dehydrogenase) were catalytically less active overall by at least an order of magnitude, due to increased
K
m
and lower
k
cat
values. Accordingly, alternative and/or bifunctional metabolic roles of these proteins in plant defense cannot be ruled out. Comprehensive analyses of lignified tissues of various
Arabidopsis
knockout mutants (for AtCAD5, 6, and 9) at different stages of growth/development indicated the presence of functionally redundant CAD metabolic networks. Moreover, disruption of AtCAD5 expression had only a small effect on either overall lignin amounts deposited, or on syringyl-guaiacyl compositions, despite being the most catalytically active form
in vitro
.
Heme regulates the dynamic exchange of Bach1 and NF-E2-related factors in the Maf transcription factor network
Authors: Sun Jiying , Brand Marjorie , Zenke Yukari , Tashiro Satoshi , Groudine Mark , Igarashi Kazuhiko
Published: February 2004

Small Maf proteins serve as dual-function transcription factors through an exchange of their heterodimerization partners. For example, as heterodimers with hematopoietic cell-specific p45 NF-E2 or NF-E2-related factors (Nrf), they activate the β-globin or antioxidative stress enzyme heme oxygenase 1 (HO-1) genes, respectively. In contrast, together with Bach1, they repress these same genes. However, the signals leading to this partner exchange are not known. Using chromatin immunoprecipitation assays in NIH 3T3 cells, we show that heme, an inducer of
ho-1
, promotes displacement of Bach1 from the MafK-occupied
ho-1
enhancers, which is followed by Nrf2 binding to these elements. Whereas histone H3 at the
ho-1
enhancers and promoter is hyperacetylated irrespective of gene activity, exposure of cells to heme results in
de novo
hyperacetylation and hypermethylation of histone H3 in the transcribed region. These data indicate that, under normal conditions, the chromatin structure of
ho-1
is in a preactivation state, but transcription is repressed by Bach1. Heme induces switching of Maf dimers, resulting in
ho-1
expression. Heme also promotes displacement of Bach1 from the β-globin locus control region without affecting MafK binding in murine erythroleukemia cells. Thus, heme functions as a signaling molecule for gene expression in higher eukaryotes.
Centrosome maturation: Measurement of microtubule nucleation throughout the cell cycle by using GFP-tagged EB1
Authors: Piehl Michelle , Tulu U. Serdar , Wadsworth Pat , Cassimeris Lynne
Published: February 2004

Understanding how cells regulate microtubule nucleation during the cell cycle has been limited by the inability to directly observe nucleation from the centrosome. To view nucleation in living cells, we imaged GFP-tagged EB1, a microtubule tip-binding protein, and determined rates of nucleation by counting the number of EB1-GFP comets emerging from the centrosome over time. Nucleation rate increased 4-fold between G
2
and prophase and continued to rise through anaphase and telophase, reaching a maximum of 7 times interphase rates. We tested several models for centrosome maturation, including γ-tubulin recruitment and increased centrosome size. The centrosomal concentration of γ-tubulin reached a maximum at metaphase, and centrosome size increased through anaphase, whereas nucleation remained high through telophase, implying the presence of additional regulatory processes. Injection of anti-γ-tubulin antibodies significantly blocked nucleation during metaphase but was less effective during anaphase, suggesting that a nucleation mechanism independent of γ-tubulin contributes to centrosome function after metaphase.
Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation
Authors: Bourne Yves , Kolb Hartmuth C. , Radić Zoran , Sharpless K. Barry , Taylor Palmer , Marchot Pascale
Published: February 2004

The 1,3-dipolar cycloaddition reaction between unactivated azides and acetylenes proceeds exceedingly slowly at room temperature. However, considerable rate acceleration is observed when this reaction occurs inside the active center gorge of acetylcholinesterase (AChE) between certain azide and acetylene reactants, attached via methylene chains to specific inhibitor moieties selective for the active center and peripheral site of the enzyme. AChE catalyzes the formation of its own inhibitor in a highly selective fashion: only a single
syn1
-triazole regioisomer with defined substitution positions and linker distances is generated from a series of reagent combinations. Inhibition measurements revealed this
syn1
-triazole isomer to be the highest affinity reversible organic inhibitor of AChE with association rate constants near the diffusion limit. The corresponding
anti1
isomer, not formed by the enzyme, proved to be a respectable but weaker inhibitor. The crystal structures of the
syn1
- and
anti1
-mouse AChE complexes at 2.45- to 2.65-Å resolution reveal not only substantial binding contributions from the triazole moieties, but also that binding of the
syn1
isomer induces large and unprecedented enzyme conformational changes not observed in the
anti1
complex nor predicted from structures of the apoenzyme and complexes with the precursor reactants. Hence, the freeze-frame reaction offers both a strategically original approach for drug discovery and a means for kinetically controlled capture, as a high-affinity complex between the enzyme and its self-created inhibitor, of a highly reactive minor abundance conformer of a fluctuating protein template.
Early onset of neoplasia in the prostate and skin of mice with tissue-specific deletion of Pten
Authors: Backman Stéphanie A. , Ghazarian Danny , So Kelvin , Sanchez Otto , Wagner Kay-Uwe , Hennighausen Lothar , Suzuki Akira , Tsao Ming-Sound , Chapman William B. , Stambolic Vuk , Mak Tak W.
Published: February 2004

PTEN
is a tumor suppressor gene mutated in various advanced human neoplasias, including glioblastomas and prostate, breast, endometrial, and kidney cancers. This tumor suppressor is a lipid phosphatase that negatively regulates cell survival and proliferation mediated by phosphatidylinositol 3-kinase/protein kinase B signaling. Using the Cre–
loxP
system, we selectively inactivated Pten in murine tissues in which the
MMTV-LTR
promoter is active, resulting in hyperproliferation and neoplastic changes in Pten-null skin and prostate. These phenotypes had early onset and were completely penetrant. Abnormalities in Pten mutant skin consisted of mild epidermal hyperplasia, whereas prostates from these mice exhibited high-grade prostatic intraepithelial neoplasia (HGPIN) that frequently progressed to focally invasive cancer. These data demonstrate that Pten is an important physiological regulator of growth in the skin and prostate. Further, the early onset of HGPIN in Pten mutant males is unique to this animal model and implicates
PTEN
mutations in the initiation of prostate cancer. Consistent with high
PTEN
mutation rates in human prostate tumors, these data indicate that PTEN is a critical tumor suppressor in this organ.
Microtransplantation of functional receptors and channels from the Alzheimer's brain to frog oocytes
Authors: Miledi R. , Dueñas Z. , Martinez-Torres A. , Kawas C. H. , Eusebi F.
Published: February 2004

About a decade ago, cell membranes from the electric organ of
Torpedo
and from the rat brain were transplanted to frog oocytes, which thus acquired functional
Torpedo
and rat neurotransmitter receptors. Nevertheless, the great potential that this method has for studying human diseases has remained virtually untapped. Here, we show that cell membranes from the postmortem brains of humans that suffered Alzheimer's disease can be microtransplanted to the plasma membrane of
Xenopus
oocytes. We show also that these postmortem membranes carry neurotransmitter receptors and voltage-operated channels that are still functional, even after they have been kept frozen for many years. This method provides a new and powerful approach to study directly the functional characteristics and structure of receptors, channels, and other membrane proteins of the Alzheimer's brain. This knowledge may help in understanding the basis of Alzheimer's disease and also help in developing new treatments.
Evidence that vaccinia virulence factor E3L binds to Z-DNA
in vivo
: Implications for development of a therapy for poxvirus infection
Authors: Kim Yang-Gyun , Lowenhaupt Ky , Oh Doo-Byoung , Kim Kyeong Kyu , Rich Alexander
Published: February 2004

The E3L gene product found in all poxviruses is required for the lethality of mice in vaccinia virus infection. Both the C-terminal region, consisting of a double-stranded RNA-binding motif, and the N-terminal region (vZ
E3L
), which is similar to the Zα family of Z-DNA-binding proteins, are required for infection. It has recently been demonstrated that the function of the N-terminal domain depends on its ability to bind Z-DNA; Z-DNA-binding domains from unrelated mammalian proteins fully complement an N-terminal deletion of E3L. Mutations that decrease affinity for Z-DNA have similar effects in decreasing pathogenicity. Compounds that block the Z-DNA-binding activity of E3L may also limit infection by the poxvirus. Here we show both an
in vitro
and an
in vivo
assay with the potential to be used in screening for such compounds. Using a conformation-specific yeast one-hybrid assay, we compared the results for Z-DNA binding of vZ
E3L
with those for human Zβ
ADAR1
, a peptide that has similarity to the Zα motif but does not bind Z-DNA, and with a mutant of hZβ
ADAR1
, which binds Z-DNA. The results suggest that this system can be used for high-throughput screening.
Estrogen receptor β inhibits 17β-estradiol-stimulated proliferation of the breast cancer cell line T47D
Authors: Ström Anders , Hartman Johan , Foster James S. , Kietz Silke , Wimalasena Jay , Gustafsson Jan-Åke
Published: February 2004

Estrogen receptor (ER) β counteracts the activity of ERα in many systems. In agreement with this, we show in this study that induced expression of ERβ in the breast cancer cell line T47D reduces 17β-estradiol-stimulated proliferation when expression of ERβ mRNA equals that of ERα. Induction of ERβ reduces growth of exponentially proliferating cells with a concomitant decrease in components of the cell cycle associated with proliferation, namely cyclin E, Cdc25A (a key regulator of Cdk2), p45
Skp2
(a key regulator of p27
Kip1
proteolysis), and an increase in the Cdk inhibitor p27
Kip1
. We also observed a reduced Cdk2 activity. These findings suggest a possible role for ERβ in breast cancer and imply that ERβ-specific ligands may reduce proliferation of ER-positive breast cancer cells through actions on the G
1
phase cell-cycle machinery.
Folate synthesis in plants: The
p
-aminobenzoate branch is initiated by a bifunctional PabA-PabB protein that is targeted to plastids
Authors: Basset Gilles J. C. , Quinlivan Eoin P. , Ravanel Stéphane , Rébeillé Fabrice , Nichols Brian P. , Shinozaki Kazuo , Seki Motoaki , Adams-Phillips Lori C. , Giovannoni James J. , Gregory Jesse F. , Hanson Andrew D.
Published: February 2004

It is not known how plants synthesize the
p
-aminobenzoate (PABA) moiety of folates. In
Escherichia coli
, PABA is made from chorismate in two steps. First, the PabA and PabB proteins interact to catalyze transfer of the amide nitrogen of glutamine to chorismate, forming 4-amino-4-deoxychorismate (ADC). The PabC protein then mediates elimination of pyruvate and aromatization to give PABA. Fungi, actinomycetes, and
Plasmodium
spp. also synthesize PABA but have proteins comprising fused domains homologous to PabA and PabB. These bipartite proteins are commonly called “PABA synthases,” although it is unclear whether they produce PABA or ADC. Genomic approaches identified
Arabidopsis
and tomato cDNAs encoding bipartite proteins containing fused PabA and PabB domains, plus a putative chloroplast targeting peptide. These cDNAs encode functional enzymes, as demonstrated by complementation of an
E. coli pabA pabB
double mutant and a yeast PABA-synthase deletant. The partially purified recombinant
Arabidopsis
protein did not produce PABA unless the
E. coli
PabC enzyme was added, indicating that it forms ADC, not PABA. The enzyme behaved as a monomer in size-exclusion chromatography and was not inhibited by physiological concentrations of PABA, its glucose ester, or folates. When the putative targeting peptide was fused to GFP and expressed in protoplasts, the fusion protein appeared only in chloroplasts, indicating that PABA synthesis is plastidial. In the pericarp of tomato fruit, the PabA-PabB mRNA level fell drastically as ripening advanced, but there was no fall in total PABA content, which stayed between 0.7 and 2.3 nmol·g
-1
fresh weight.
Diet, nutrition and the prevention of type 2 diabetes
Authors: Steyn NP , Mann J , Bennett PH , Temple N , Zimmet P , Tuomilehto J , Lindström J , Louheranta A
Published: February 2004

Objectives:

The overall objective of this study was to evaluate and provide evidence and recommendations on current published literature about diet and lifestyle in the prevention of type 2 diabetes.

Design:

Epidemiological and experimental studies, focusing on nutritional intervention in the prevention of type 2 diabetes are used to make disease-specific recommendations. Long-term cohort studies are given the most weight as to strength of evidence available.

Setting and subjects:

Numerous clinical trials and cohort studies in low, middle and high income countries are evaluated regarding recommendations for dietary prevention of type 2 diabetes. These include, among others, the Finnish Diabetes Prevention Study, US Diabetes Prevention Program, Da Qing Study; Pima Indian Study; Iowa Women's Health Study; and the study of the US Male Physicians.

Results:

There is convincing evidence for a decreased risk of diabetes in adults who are physically active and maintain a normal body mass index (BMI) throughout adulthood, and in overweight adults with impaired glucose tolerance who lose weight voluntarily. An increased risk for developing type 2 diabetes is associated with overweight and obesity; abdominal obesity; physical inactivity; and maternal diabetes. It is probable that a high intake of saturated fats and intrauterine growth retardation also contribute to an increased risk, while non-starch polysaccharides are likely to be associated with a decreased risk. From existing evidence it is also possible that omega-3 fatty acids, low glycaemic index foods and exclusive breastfeeding may play a protective role, and that total fat intake andtransfatty acids may contribute to the risk. However, insufficient evidence is currently available to provide convincing proof.

Conclusions:

Based on the strength of available evidence regarding diet and lifestyle in the prevention of type 2 diabetes, it is recommended that a normal weight status in the lower BMI range (BMI 21–23) and regular physical activity be maintained throughout adulthood; abdominal obesity be prevented; and saturated fat intake be less than 7% of the total energy intake.
Diet, nutrition and the prevention of dental diseases
Authors: Moynihan Paula , Petersen Poul Erik
Published: February 2004

Oral health is related to diet in many ways, for example, nutritional influences on craniofacial development, oral cancer and oral infectious diseases. Dental diseases impact considerably on self-esteem and quality of life and are expensive to treat. The objective of this paper is to review the evidence for an association between nutrition, diet and dental diseases and to present dietary recommendations for their prevention. Nutrition affects the teeth during development and malnutrition may exacerbate periodontal and oral infectious diseases. However, the most significant effect of nutrition on teeth is the local action of diet in the mouth on the development of dental caries and enamel erosion. Dental erosion is increasing and is associated with dietary acids, a major source of which is soft drinks.

Despite improved trends in levels of dental caries in developed countries, dental caries remains prevalent and is increasing in some developing countries undergoing nutrition transition. There is convincing evidence, collectively from human intervention studies, epidemiological studies, animal studies and experimental studies, for an association between the amount and frequency of free sugars intake and dental caries. Although other fermentable carbohydrates may not be totally blameless, epidemiological studies show that consumption of starchy staple foods and fresh fruit are associated with low levels of dental caries. Fluoride reduces caries risk but has not eliminated dental caries and many countries do not have adequate exposure to fluoride.

It is important that countries with a low intake of free sugars do not increase intake, as the available evidence shows that when free sugars consumption is <15–20kg/yr (~6–10% energy intake), dental caries is low. For countries with high consumption levels it is recommended that national health authorities and decision-makers formulate country-specific and community-specific goals for reducing the amount of free sugars aiming towards the recommended maximum of no more than 10% of energy intake. In addition, the frequency of consumption of foods containing free sugars should be limited to a maximum of 4 times per day. It is the responsibility of national authorities to ensure implementation of feasible fluoride programmes for their country.
In Candida albicans, the Nim1 kinases Gin4 and Hsl1 negatively regulate pseudohypha formation and Gin4 also controls septin organization
Authors: Wightman Raymond , Bates Steven , Amornrrattanapan Pat , Sudbery Peter
Published: February 2004

In the development of hyphal germ tubes of Candida albicans, a band of septin forms at the base of the germ tube (basal septin band). Later, a septin ring forms, which organizes the first septum within the germ tube (septin ring). We have investigated the role of the Nim1 kinases, Gin4 and Hsl1, in the formation of these septin structures. We show that during germ tube formation, Gin4 is required for the organization of the septin ring but not the basal septin band. Hsl1 is not required for the formation of either septin rings or basal bands. Unexpectedly, we found that both gin4Δ and hsl1Δ mutants form pseudohyphae constitutively, in a fashion that in the case of gin4Δ, is partly independent of Swe1. Gin4-depleted pseudohyphae are unable to form hyphae when challenged with serum, but this can be overcome by ectopic expression of Gin4 from the MET3 promoter. Thus, Gin4 may regulate the developmental switch from pseudohyphae to hyphae.
Spindle checkpoint proteins and chromosome–microtubule attachment in budding yeast
Authors: Gillett Emily S. , Espelin Christopher W. , Sorger Peter K.
Published: February 2004

Accurate chromosome segregation depends on precise regulation of mitosis by the spindle checkpoint. This checkpoint monitors the status of kinetochore–microtubule attachment and delays the metaphase to anaphase transition until all kinetochores have formed stable bipolar connections to the mitotic spindle. Components of the spindle checkpoint include the mitotic arrest defective (MAD) genes MAD1–3, and the budding uninhibited by benzimidazole (BUB) genes BUB1 and BUB3. In animal cells, all known spindle checkpoint proteins are recruited to kinetochores during normal mitoses. In contrast, we show that whereas Saccharomyces cerevisiae Bub1p and Bub3p are bound to kinetochores early in mitosis as part of the normal cell cycle, Mad1p and Mad2p are kinetochore bound only in the presence of spindle damage or kinetochore lesions that interfere with chromosome–microtubule attachment. Moreover, although Mad1p and Mad2p perform essential mitotic functions during every division cycle in mammalian cells, they are required in budding yeast only when mitosis goes awry. We propose that differences in the behavior of spindle checkpoint proteins in animal cells and budding yeast result primarily from evolutionary divergence in spindle assembly pathways.
Mice lacking JunB are osteopenic due to cell-autonomous osteoblast and osteoclast defects
Authors: Kenner Lukas , Hoebertz Astrid , Beil F. Timo , Keon Niamh , Karreth Florian , Eferl Robert , Scheuch Harald , Szremska Agnieszka , Amling Michael , Schorpp-Kistner Marina , Angel Peter , Wagner Erwin F.
Published: February 2004

Because JunB is an essential gene for placentation, it was conditionally deleted in the embryo proper. JunBΔ/Δ mice are born viable, but develop severe low turnover osteopenia caused by apparent cell-autonomous osteoblast and osteoclast defects before a chronic myeloid leukemia-like disease. Although JunB was reported to be a negative regulator of cell proliferation, junBΔ/Δ osteoclast precursors and osteoblasts show reduced proliferation along with a differentiation defect in vivo and in vitro. Mutant osteoblasts express elevated p16INK4a levels, but exhibit decreased cyclin D1 and cyclin A expression. Runx2 is transiently increased during osteoblast differentiation in vitro, whereas mature osteoblast markers such as osteocalcin and bone sialoprotein are strongly reduced. To support a cell-autonomous function of JunB in osteoclasts, junB was inactivated specifically in the macrophage–osteoclast lineage. Mutant mice develop an osteopetrosis-like phenotype with increased bone mass and reduced numbers of osteoclasts. Thus, these data reveal a novel function of JunB as a positive regulator controlling primarily osteoblast as well as osteoclast activity.
Quantitation of mitochondrial dynamics by photolabeling of individual organelles shows that mitochondrial fusion is blocked during the Bax activation phase of apoptosis
Authors: Karbowski Mariusz , Arnoult Damien , Chen Hsiuchen , Chan David C. , Smith Carolyn L. , Youle Richard J.
Published: February 2004

A dynamic balance of organelle fusion and fission regulates mitochondrial morphology. During apoptosis this balance is altered, leading to an extensive fragmentation of the mitochondria. Here, we describe a novel assay of mitochondrial dynamics based on confocal imaging of cells expressing a mitochondrial matrix–targeted photoactivable green fluorescent protein that enables detection and quantification of organelle fusion in living cells. Using this assay, we visualize and quantitate mitochondrial fusion rates in healthy and apoptotic cells. During apoptosis, mitochondrial fusion is blocked independently of caspase activation. The block in mitochondrial fusion occurs within the same time range as Bax coalescence on the mitochondria and outer mitochondrial membrane permeabilization, and it may be a consequence of Bax/Bak activation during apoptosis.
Activation of Virus-specific Memory B Cells in the Absence of T Cell Help
Authors: Hebeis Barbara J. , Klenovsek Karin , Rohwer Peter , Ritter Uwe , Schneider Andrea , Mach Michael , Winkler Thomas H.
Published: February 2004

Humoral immunity is maintained by long-lived plasma cells, constitutively secreting antibodies, and nonsecreting resting memory B cells that are rapidly reactivated upon antigen encounter. The activation requirements for resting memory B cells, particularly the role of T helper cells, are unclear. To analyze the activation of memory B cells, mice were immunized with human cytomegalovirus, a complex human herpesvirus, and tick-born encephalitis virus, and a simple flavivirus. B cell populations devoid of Ig-secreting plasma cells were adoptively transferred into T and B cell–deficient RAG-1−/− mice. Antigenic stimulation 4–6 d after transfer of B cells resulted in rapid IgG production. The response was long lasting and strictly antigen specific, excluding polyclonal B cell activation. CD4+ T cells were not involved since (a) further depletion of CD4+ T cells in the recipient mice did not alter the antibody response and (b) recipient mice contained no detectable CD4+ T cells 90 d posttransfer. Memory B cells could not be activated by a soluble viral protein without T cell help. Transfer of memory B cells into immunocompetent animals indicated that presence of helper T cells did not enhance the memory B cell response. Therefore, our results indicate that activation of virus-specific memory B cells to secrete IgG is independent of cognate or bystander T cell help.
Developmental Separation of V(D)J Recombinase Expression and Initiation of IgH Recombination in B Lineage Progenitors In Vivo
Authors: Borghesi Lisa , Gerstein Rachel M.
Published: February 2004

In B lineage progenitors, V(D)J recombination occurs only during distinct stages of development and is restricted to immunoglobulin loci. This process is thought to be controlled by both regulated expression of the V(D)J recombinase and by limited accessibility of target loci to the recombinase complex. However, it is unknown whether these two processes occur concomitantly in developing B lineage progenitors or whether these events are temporally distinct and, therefore, potentially independently regulated. To distinguish between these possibilities, we developed a transgenic V(D)J recombination substrate that is not governed by the same chromatin remodeling constraints as endogenous immunoglobulin heavy chain (IgH) loci and examined the requirements for V(D)J recombination to initiate in early B lineage progenitors. We find that single B lineage precursors express an active V(D)J recombinase in vivo before the stage when IgH rearrangements are frequently detectable. Our results indicate that the onset of recombinase activity and the initiation of IgH recombination are developmentally distinct events in the B lineage.
Mechanism of Ivermectin Facilitation of Human P2X4 Receptor Channels
Authors: Priel Avi , Silberberg Shai D.
Published: February 2004

Ivermectin (IVM), a widely used antiparasitic agent in human and veterinary medicine, was recently shown to augment macroscopic currents through rat P2X4 receptor channels (Khakh, B.S., W.R. Proctor, T.V. Dunwiddie, C. Labarca, and H.A. Lester. 1999. J. Neurosci. 19:7289–7299.). In the present study, the effects of IVM on the human P2X4 (hP2X4) receptor channel stably transfected in HEK293 cells were investigated by recording membrane currents using the patch clamp technique. In whole-cell recordings, IVM (≤10 μM) applied from outside the cell (but not from inside) increased the maximum current activated by ATP, and slowed the rate of current deactivation. These two phenomena likely result from the binding of IVM to separate sites. A higher affinity site (EC50 0.25 μM) increased the maximal current activated by saturating concentrations of ATP without significantly changing the rate of current deactivation or the EC50 and Hill slope of the ATP concentration-response relationship. A lower affinity site (EC50 2 μM) slowed the rate of current deactivation, and increased the apparent affinity for ATP. In cell-attached patch recordings, P2X4 receptor channels exhibited complex kinetics, with multiple components in both the open and shut distributions. IVM (0.3 μM) increased the number of openings per burst, without significantly changing the mean open or mean shut time within a burst. At higher concentrations (1.5 μM) of IVM, two additional open time components of long duration were observed that gave rise to long-lasting bursts of channel activity. Together, the results suggest that the binding of IVM to the higher affinity site increases current amplitude by reducing channel desensitization, whereas the binding of IVM to the lower affinity site slows the deactivation of the current predominantly by stabilizing the open conformation of the channel.
Impaired p53 Expression, Function, and Nuclear Localization in Calreticulin-deficient Cells
Authors: Mesaeli Nasrin , Phillipson Clark
Published: February 2004

The tumor suppressor protein, p53 is a transcription factor that not only activates expression of genes containing the p53 binding site but also can repress the expression of some genes lacking this binding site. Previous studies have shown that overexpression of wild-type p53 leads to apoptosis and cell cycle arrest. DNA damage, such as that caused by UV irradiation, results in p53 stabilization and nuclear localization that subsequently induces apoptosis. Recently, the level of calreticulin (CRT) has been correlated with the rate of apoptosis. Therefore, the aim of this study was to investigate the role of CRT in the regulation of apoptosis via modulating p53 function and expression. Here we show a significant decrease in both basal and DNA damage induced p53 functions in the CRT-deficient cells (crt-/-). This study is the first to demonstrate that CRT function is required for the stability and localization of the p53 protein. By using immuonocytochemical techniques, we showed that observed changes in p53 in the crt-/- cells are due to the nuclear accumulation of Mdm2 (murine double minute gene). These results, lead us to conclude that CRT regulates p53 function by affecting its rate of degradation and nuclear localization.
Mmd1p, a Novel, Conserved Protein Essential for Normal Mitochondrial Morphology and Distribution in the Fission YeastSchizosaccharomyces pombe
Authors: Weir Barbara A. , Yaffe Michael P.
Published: February 2004

The mmd1 mutation causes temperature-sensitive growth and defects in mitochondrial morphology and distribution in the fission yeast Schizosaccharomyces pombe. In mutant cells, mitochondria aggregate at the two cell ends, with increased aggregation at elevated temperatures. Microtubules, which mediate mitochondrial positioning in fission yeast, seem normal in mmd1 cells at permissive temperature and after several hours at the nonpermissive temperature but display aberrant organization after prolonged periods at 37°C. Additionally, cells harboring both mmd1 and ban5-4, a temperature-sensitive allele of α2-tubulin, display synthetic defects in growth and mitochondrial distribution. The mmd1 mutation maps to an open reading frame encoding a novel 35.7-kDa protein. The Mmd1p sequence features repeating EZ-HEAT motifs and displays high conservation with uncharacterized homologues found in a variety of organisms. Saccharomyces cerevisiae cells depleted for their MMD1 homologue show increased sensitivity to the antimicrotubule drug benomyl, and the S. cerevisiae gene complemented the S. pombe mutation. Mmd1p was localized to the cytosol. Mmd1p is the first identified component required for the alignment of mitochondria along microtubules in fission yeast.