bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective involvement of TIMP‐2 in the second activational cleavage of pro‐MMP‐2: refinement of the pro‐MMP‐2 activation mechanism
Authors: Lafleur Marc A , Tester Angus M , Thompson Erik W
Published: October 2003

A tissue inhibitor of metalloproteinases‐2 (TIMP‐2)‐independent mechanism for generating the first activational cleavage of pro‐matrix metalloproteinase‐2 (MMP‐2) was identified in membrane type‐1 MMP (MT1‐MMP)‐transfected MCF‐7 cells and confirmed in TIMP‐2‐deficient fibroblasts. In contrast, the second MMP‐2‐activational step was found to be TIMP‐2 dependent in both systems. MMP‐2 hemopexin C‐terminal domain was found to be critical for the first step processing, confirming a need for membrane tethering. We propose that the intermediate species of MMP‐2 forms the well‐established trimolecular complex (MT1‐MMP/TIMP‐2/MMP‐2) for further TIMP‐2‐dependent autocatalytic cleavage to the fully active species. This alternate mechanism may supplement the traditional TIMP‐2‐mediated first step mechanism.
Potassium channels
Authors: MacKinnon Roderick
Published: October 2003

The atomic structures of K+ channels have added a new dimension to our understanding of K+ channel function. I will briefly review how structures have influenced our views on ion conduction, gating of the pore, and voltage sensing.
Sierra Leone – investing in nutrition to reduce poverty: a call for action
Authors: Aguayo Victor M , Scott Sylvetta , Ross Jay
Published: October 2003

Background:

Malnutrition rates in Sierra Leone are among the highest in the world. However, policy-makers do not always recognise the fight against malnutrition as a policy priority to ensure the healthy human capital needed to fight poverty and achieve sustained positive economic growth.

Objective:

The analysis presented here was conducted by an intersectoral and inter-agency group of Sierra Leonean senior policy advisors to quantify some of the potential human and economic benefits of improved policies and programmes to reduce malnutrition.

Findings:

The analysis revealed that 46% of child deaths in Sierra Leone are attributable to malnutrition, the single greatest cause of child mortality in the country. In the absence of adequate policy and programme action, malnutrition will be the underlying cause of an estimated 74000 child deaths over the next five years. The analysis also revealed that if current levels of iodine deficiency remain unchanged over the next five years, 252000 children could be born with varying degrees of mental retardation as a result of intrauterine iodine deficiency. Finally, the analysis showed that, in the absence of adequate policy and programme action to reduce the unacceptable rates of anaemia in women, the monetary value of agricultural productivity losses associated with anaemia in the female labour force over the next five years will exceed $94.5 million.

Conclusion:

Sustained investment in nutrition in Sierra Leone could bring about enormous human and economic benefits to develop the social sector, revitalise the economy, and attain the poverty reduction goals that Sierra Leone has set forth.
De Novo Amino Acid Biosynthesis in Potato Tubers Is Regulated by Sucrose Levels
Authors: Roessner-Tunali Ute , Urbanczyk-Wochniak Ewa , Czechowski Tomasz , Kolbe Anna , Willmitzer Lothar , Fernie Alisdair R.
Published: October 2003

Plant growth and development are strongly dependent on sink-source interactions. In the majority of plants, sucrose (Suc) is the dominant form in which photo-assimilate is transported from source to sinks. Although the effects of Suc on photosynthetic metabolism have been intensively studied, the effect of Suc supply on metabolism in sink organs has received relatively little attention. For this reason, we performed a detailed characterization of the metabolism of potato (Solanum tuberosum) plants in which the Suc supply to the tuber was restricted by genetic or environmental perturbation. These characterizations revealed a clear inverse relationship between the levels of Suc and free amino acids. When data obtained from this study were considered alongside our previous work, a negative correlation between tuber Suc and amino acid content became apparent. Analysis of the transcript levels of key enzymes involved in amino acid biosynthesis revealed that several of these were increased under these conditions. Taken together, these data strongly suggest that Suc regulates amino acid biosynthesis in storage tissues such as potato tubers, most probably at the level of transcription.
The G-Protein-Coupled Receptor GCR1 Regulates DNA Synthesis through Activation of Phosphatidylinositol-Specific Phospholipase C
Authors: Apone Fabio , Alyeshmerni Nicole , Wiens Kathryn , Chalmers Derek , Chrispeels Maarten J. , Colucci Gabriella
Published: October 2003

Different lines of evidence suggest that specific events during the cell cycle may be mediated by a heterotrimeric G-protein activated by a cognate G-protein coupled receptor. However, coupling between the only known Gα-subunit of the heterotrimeric G-protein (GPA1) and the only putative G-protein coupled receptor (GCR1) of plants has never been shown. Using a variety of approaches, we show here that GCR1-enhanced thymidine incorporation into DNA depends on an increase in phosphatidylinositol-specific phospholipase C activity and an elevation of inositol 1,4,5-trisphosphate levels in the cells. Tobacco (Nicotiana tabacum) cells that overexpress either Arabidopsis GCR1 or GPA1 display this phenomenon. We suggest on the basis of these results that GCR1-controlled events during the cell cycle involve phosphatidylinositol-specific phospholipase C as an effector of GCR1 and inositol 1,4,5-trisphosphate as a second messenger, and that GCR1 and GPA1 are both involved in this particular signaling pathway.
Proton Transport in Maize Tonoplasts Supported by Fructose-1,6-Bisphosphate Cleavage. Pyrophosphate-Dependent Phosphofructokinase as a Pyrophosphate-Regenerating System
Authors: Costa dos Santos Anelise , Seixas da-Silva Wagner , de Meis Leopoldo , Galina Antonio
Published: October 2003

The energy derived from pyrophosphate (PPi) hydrolysis is used to pump protons across the tonoplast membrane, thus forming a proton gradient. In a plant's cytosol, the concentration of PPi varies between 10 and 800 μm, and the PPi concentration needed for one-half maximal activity of the maize (Zea mays) root tonoplast H+-pyrophosphatase is 30 μm. In this report, we show that the H+-pyrophosphatase of maize root vacuoles is able to hydrolyze PPi (Reaction 2) formed by Reaction 1, which is catalyzed by PPi-dependent phosphofructokinase (PFP):  Fructose-1,6-bisphosphate (F1,6BP) + Pi ↔ PPi +Fructose-6-phosphate (F6 P)(reaction 1)PPi → 2 Pi(reaction 2)H+  cyt → H+  vac  (reaction 3) F1,6BP + H+  cyt ↔ H+  vac + F6P + Pi(reaction 4) During the steady state, one-half of the inorganic phosphate released (Reaction 4) is ultimately derived from F1,6BP, whereas PFP continuously regenerates the pyrophosphate (PPi) hydrolyzed. A proton gradient (ΔpH) can be built up in tonoplast vesicles using PFP as a PPi-regenerating system. The Δ pH formed by the H+-pyrophosphatase can be dissipated by addition of 20 mm F6P, which drives Reaction 1 to the left and decreases the PPi available for the H+-pyrophosphatase. The maximal Δ pH attained by the pyrophosphatase coupled to the PFP reaction can be maintained by PFP activities far below those found in higher plants tissues.
An ATP-Binding Cassette Transporter GhWBC1 from Elongating Cotton Fibers
Authors: Zhu Yong-Qing , Xu Ke-Xiang , Luo Bin , Wang Jia-Wei , Chen Xiao-Ya
Published: October 2003

We have isolated a cDNA (GhWBC1) from cotton (Gossypium hirsutum) that encodes an ATP-binding cassette transporter of the WBC (white/brown complex) subfamily. Members of this subfamily are half-sized transporters and are reported to mediate lipid and drug excretion in human (Homo sapiens). GhWBC1 is highly expressed in developing fiber cells, but transcripts were also detectable in other tissues except roots. The transcript level peaked in rapidly expanding fibers from 5 to 9 DPA and then decreased. The GhWBC1 expression was weak in fiber cells of an li (ligon-lintless) mutant, which is defective in fiber cell elongation. These data indicate that GhWBC1 gene expression correlates with cotton fiber elongation. Transient expression of enhanced green fluorescence protein-GhWBC1 fusion protein in onion (Allium cepa) epidermal cells revealed plasma membrane localization. The GhWBC1 cDNA driven by a constitutive 35S promoter was introduced into Arabidopsis. About 13% of the transformants produced short siliques (SSs), whereas others had normal siliques (long siliques [LSs]). In siliques of SS lines, most embryos were severely shriveled, and only several seeds per silique could be found at maturity. The transgene expression level was higher in SS lines than in LS lines. Expression of AtWBC11, the closest homolog of GhWBC1 in Arabidopsis, was not altered in either SS or LS transgenic plants examined. These data suggest that GhWBC1 interferes with substance translocation that is required for Arabidopsis seed and silique development. Characterization of Arabidopsis WBC members, particularly AtWBC11, will help to dissect the role of GhWBC1 in cotton fiber development and elongation.
Cold Induction of Arabidopsis CBF Genes Involves Multiple ICE (Inducer of CBF Expression) Promoter Elements and a Cold-Regulatory Circuit That Is Desensitized by Low Temperature
Authors: Zarka Daniel G. , Vogel Jonathan T. , Cook Daniel , Thomashow Michael F.
Published: October 2003

The Arabidopsis CBF1, 2, and 3 genes (also known as DREB1b, c, and a, respectively) encode transcriptional activators that have a central role in cold tolerance. CBF1-3 are rapidly induced upon exposing plants to low temperature, followed by expression of CBF-targeted genes, the CBF regulon, resulting in an increase in plant freezing tolerance. At present, little is known about the cold-sensing mechanism that controls CBF expression. Results presented here indicate that this mechanism does not require a cold shock to bring about the accumulation of CBF transcripts, but instead, absolute temperature is monitored with a greater degree of input, i.e. lower temperature, resulting in a greater output, i.e. higher levels of CBF transcripts. Temperature-shift experiments also indicate that the cold-sensing mechanism becomes desensitized to a given low temperature, such as 4°C, and that resensitization to that temperature requires between 8 and 24 h at warm temperature. Gene fusion experiments identified a 125-bp section of the CBF2 promoter that is sufficient to impart cold-responsive gene expression. Mutational analysis of this cold-responsive region identified two promoter segments that work in concert to impart robust cold-regulated gene expression. These sequences, designated ICEr1 and ICEr2 (induction of CBF expression region 1 or 2), were also shown to stimulate transcription in response to mechanical agitation and the protein synthesis inhibitor, cycloheximide.
Characterization of the Targeting Signal of the Arabidopsis 22-kD Integral Peroxisomal Membrane Protein
Authors: Murphy Mary A. , Phillipson Belinda A. , Baker Alison , Mullen Robert T.
Published: October 2003

Using a combination of in vivo and in vitro assays, we characterized the sorting pathway and molecular targeting signal for the Arabidopsis 22-kD peroxisome membrane protein (PMP22), an integral component of the membrane of all peroxisomes in the mature plant. We show that nascent PMP22 is sorted directly from the cytosol to peroxisomes and that it is inserted into the peroxisomal boundary membrane with its N- and C-termini facing the cytosol. This direct sorting of PMP22 to peroxisomes contrasts with the indirect sorting reported previously for cottonseed (Gossypium hirsutum) ascorbate peroxidase, an integral PMP that sorts to peroxisomes via a subdomain of the endoplasmic reticulum. Thus, at least two different sorting pathways for PMPs exist in plant cells. At least four distinct regions within the N-terminal one-half of PMP22, including a positively charged domain present in most peroxisomal integral membrane-destined proteins, functions in a cooperative manner in efficient peroxisomal targeting and insertion. In addition, targeting with high fidelity to peroxisomes requires all four membrane-spanning domains in PMP22. Together, these results illustrate that the PMP22 membrane peroxisomal targeting signal is complex and that different elements within the signal may be responsible for mediating unique aspects of PMP22 biogenesis, including maintaining the solubility before membrane insertion, targeting to peroxisomes, and ensuring proper assembly in the peroxisomal boundary membrane.
A Negative Regulatory Element Controls mRNA Abundance of the
Leishmania mexicana
Paraflagellar Rod Gene
PFR2
Authors: Mishra Krishna K. , Holzer Timothy R. , Moore Landon L. , LeBowitz Jonathan H.
Published: October 2003

The
Leishmania mexicana
PFR2 locus encodes a component of the paraflagellar rod (PFR), a flagellar structure found only in the insect stage of the life cycle.
PFR2
mRNA levels are 10-fold lower in the mammalian stage than in the insect stage. Nuclear run-on experiments indicate that the change in
PFR2
mRNA abundance is achieved posttranscriptionally. Deletion and block substitution analysis of the entire 1,400-nucleotide 3′ untranslated region (UTR) of
PFR2C
led to the identification of a regulatory element contained within 10 nucleotides of the 3′ UTR, termed the PFR regulatory element (PRE), that is necessary for the 10-fold regulation of
PFR2
mRNA levels. Comparison of the half-lives of
PFR2
transcripts, identical except for the presence or absence of the PRE, revealed that the PRE acts by destabilizing the
PFR2
mRNA in amastigotes. The PRE was inserted into a construct which directs the constitutive expression of a chimeric
PFR2
transcript. Insertion of the PRE resulted in regulated expression of this transcript, demonstrating that the regulatory element is sufficient for promastigote-specific expression. Since the PRE is present in the 3′ UTR of all
L. mexicana
PFR genes examined so far, we propose that it serves a means of coordinating expression of PFR genes.
Recapitulation of the Sexual Cycle of the Primary Fungal Pathogen
Cryptococcus neoformans
var.
gattii
: Implications for an Outbreak on Vancouver Island, Canada
Authors: Fraser James A. , Subaran Ryan L. , Nichols Connie B. , Heitman Joseph
Published: October 2003

Cryptococcus neoformans
is a human fungal pathogen that exists as three distinct varieties or sibling species: the predominantly opportunistic pathogens
C. neoformans
var.
neoformans
(serotype D) and
C. neoformans
var.
grubii
(serotype A) and the primary pathogen
C. neoformans
var.
gattii
(serotypes B and C). While serotypes A and D are cosmopolitan, serotypes B and C are typically restricted to tropical regions. However, serotype B isolates of
C. neoformans
var.
gattii
have recently caused an outbreak on Vancouver Island in Canada, highlighting the threat of this fungus and its capacity to infect immunocompetent individuals. Here we report a large-scale analysis of the mating abilities of serotype B and C isolates from diverse sources and identify unusual strains that mate robustly and are suitable for further genetic analysis. Unlike most isolates, which are of both the
a
and α mating types but are predominantly sterile, the majority of the Vancouver outbreak strains are exclusively of the α mating type and the majority are fertile. In an effort to enhance mating of these isolates, we identified and disrupted the
CRG1
gene encoding the GTPase-activating protein involved in attenuating pheromone response.
crg1
mutations dramatically increased mating efficiency and enabled mating with otherwise sterile isolates. Our studies provide a genetic and molecular foundation for further studies of this primary pathogen and reveal that the Vancouver Island outbreak may be attributable to a recent recombination event.
Developmentally Regulated Chromosome Fragmentation Linked to Imprecise Elimination of Repeated Sequences in Paramecia
Authors: Le Mouël Anne , Butler Alain , Caron François , Meyer Eric
Published: October 2003

The chromosomes of ciliates are fragmented at reproducible sites during the development of the polyploid somatic macronucleus, but the mechanisms involved appear to be quite diverse in different species. In
Paramecium aurelia
, the process is imprecise and results in de novo telomere addition at locally heterogeneous positions. To search for possible determinants of chromosome fragmentation, we have studied an ∼21-kb fragmentation region from the germ line genome of
P. primaurelia
. The mapping and sequencing of alternative macronuclear versions of the region show that two distinct multicopy elements, a minisatellite and a degenerate transposon copy, are eliminated by an imprecise mechanism leading either to chromosome fragmentation and the formation of new telomeres or to the rejoining of flanking sequences. Heterogeneous internal deletions occur between short direct repeats containing TA dinucleotides. The complex rearrangement patterns produced vary slightly among genetically identical cell lines, show non-Mendelian inheritance during sexual reproduction, and can be experimentally modified by transformation of the maternal macronucleus with homologous sequences. These results suggest that chromosome fragmentation in
Paramecium
is the consequence of imprecise DNA elimination events that are distinct from the precise excision of single-copy internal eliminated sequences and that target multicopy germ line sequences by homology-dependent epigenetic mechanisms.
Termite Gut Symbiotic Archaezoa Are Becoming Living Metabolic Fossils
Authors: Li Li , Fröhlich Jürgen , Pfeiffer Peter , König Helmut
Published: October 2003

Over the course of several million years, the eukaryotic gut symbionts of lower termites have become adapted to a cellulolytic environment. Up to now it has been believed that they produce nutriments using their own cellulolytic enzymes for the benefit of their termite host. However, we have now isolated two endoglucanases with similar apparent molecular masses of approximately 36 kDa from the not yet culturable symbiotic Archaezoa living in the hindgut of the most primitive Australian termite,
Mastotermes darwiniensis
. The N-terminal sequences of these cellulases exhibited significant homology to cellulases of termite origin, which belong to glycosyl hydrolase family 9. The corresponding genes were detected not in the mRNA pool of the flagellates but in the salivary glands of
M. darwiniensis
. This showed that cellulases isolated from the flagellate cells originated from the termite host. By use of a PCR-based approach, DNAs encoding cellulases belonging to glycosyl hydrolase family 45 were obtained from micromanipulated nuclei of the flagellates
Koruga bonita
and
Deltotrichonympha nana
. These results indicated that the intestinal flagellates of
M. darwiniensis
take up the termite's cellulases from gut contents.
K. bonita
and
D. nana
possess at least their own endoglucanase genes, which are still expressed, but without significant enzyme activity in the nutritive vacuole. These findings give the impression that the gut Archaezoa are heading toward a secondary loss of their own endoglucanases and that they use exclusively termite cellulases.
Biosynthesis of Glycosylphosphatidylinositol Is Essential to the Survival of the Protozoan Parasite
Toxoplasma gondii
Authors: Wichroski Michael J. , Ward Gary E.
Published: October 2003

The
PIGA
gene from
Toxoplasma gondii
has been cloned and characterized. Like mammalian PIGA, the transmembrane and C-terminal domains are sufficient to direct localization to the parasite endoplasmic reticulum. A functional copy of
PIGA
is required for tachyzoite viability, demonstrating that glycosylphosphatidylinositol biosynthesis is an essential process in
T. gondii.
α-Pheromone-Induced “Shmooing” and Gene Regulation Require White-Opaque Switching during
Candida albicans
Mating
Authors: Lockhart Shawn R. , Zhao Rui , Daniels Karla J. , Soll David R.
Published: October 2003

A 14-mer α-pheromone peptide of
Candida albicans
was chemically synthesized and used to analyze the role of white-opaque switching in the mating process. The α-pheromone peptide blocked cell multiplication and induced “shmooing” in
a/a
cells expressing the opaque-phase phenotype but not in
a/a
cells expressing the white-phase phenotype. The α-pheromone peptide induced these effects at 25°C but not at 37°C. An analysis of mating-associated gene expression revealed several categories of gene regulation, including (i)
MTL
-homozygous-specific, pheromone stimulated, switching-independent (
CAG1
and
STE4
); (ii) mating type-specific, pheromone-induced, switching-independent (
STE2
); and (iii) pheromone-induced, switching-dependent (
FIG1
,
KAR4
, and
HWP1
). An analysis of switching-regulated genes revealed an additional category of opaque-phase-specific genes that are downregulated by α-pheromone only in
a/a
cells (
OP4
,
SAP1
, and
SAP3
). These results demonstrate that α-pheromone causes shmooing, the initial step in the mating process, only in
a/a
cells expressing the opaque phenotype and only at temperatures below that in the human host. These results further demonstrate that although some mating-associated genes are stimulated by the α-pheromone peptide in both white- and opaque-phase cells, others are stimulated only in opaque-phase cells, revealing a category of gene regulation unique to
C. albicans
in which α-pheromone induction requires the white-opaque transition. These results demonstrate that in
C. albicans
, the mating process and associated gene regulation must be examined within the context of white-opaque switching.
Deficiency of mannose 6‐phosphate receptors and lysosomal storage: a morphometric analysis of hepatocytes of neonatal mice
Authors: Schellens Jacques P.M , Saftig Paul , Von Figura Kurt , Everts Vincent
Published: October 2003

Transport of lysosomal enzymes is mediated by two mannose 6‐phosphate receptors: a cation dependent (CD‐MPR) and a cation independent receptor (CI‐MPR). In the present study the effect of MPR‐deficiency on the lysosomal system of neonatal mouse hepatocytes was studied by ultrastructural morphometric analyses.

The volume density of the lysosomal system in hepatocytes of mice that lack both receptors was significantly increased in comparison with controls and with mice deficient for CI‐MPR only. This higher volume density was due to a nine‐fold increase of residual bodies. In CI‐MPR‐deficient mice the volume density of the lysosomal system was not different from controls and no increase of residual bodies was observed.

It is concluded that in hepatocytes of MPR‐deficient neonatal mice lysosomal storage occurs when both MPRs are lacking, whereas deficiency of CI‐MPR only has no effect on the ultrastructure of the lysosomal system.
Group Incentives and Performance: A Study of Spontaneous Goal Setting, Goal Choice and Commitment
Authors: Guthrie James P. , Hollensbe Elaine C.
Published: October 2003

Building on previous results at the individual level, this study tested the differential effects of three forms of group incentives on aspects of group goal setting, and group performance. Relationships among group incentives, spontaneous goal setting, chosen group goal level, goal commitment, and group performance were examined. Results indicate that placing pay at risk increased the level of spontaneous group goal setting. Also, groups with the most pay at risk chose higher goals. For these groups, chosen group goal level mediated the group incentives-group performance relationship.
Regulation of cell adhesion signaling by synthetic glycopolymer matrix in primary cultured hepatocyte
Authors: Kim Sang-Heon , Kim Jong-Hun , Akaike Toshihiro
Published: October 2003

Control of cell–matrix interactions is a central principle for the design of biomaterial in tissue engineering. In this study, we evaluated a synthetic glycopolymer, which is recognized by the asialoglycoprotein receptor (ASGPR) expressed on the surface of hepatocytes, as an artificial matrix to regulate integrin‐mediated signaling. The phosphorylation of focal adhesion kinase was restricted in hepatocytes cultured on the glycopolymer compared with fibronectin. In addition, there was no reorganization of cytoskeleton‐related proteins such as actin filaments, microtubules, and vinculin in hepatocytes cultured on the glycopolymer. DNA synthesis and cyclin D1 expression were suppressed in hepatocytes grown on the glycopolymer as compared with those grown on fibronectin and collagen. The data suggest that the glycopolymer will be a good artificial matrix to regulate integrin‐mediated signaling and cell growth through the unique ASGPR–carbohydrate interaction.
Enzymatic nitration of phytophenolics: evidence for peroxynitrite‐independent nitration of plant secondary metabolites
Authors: Sakihama Yasuko , Tamaki Ryoko , Shimoji Hisashi , Ichiba Toshio , Fukushi Yukiharu , Tahara Satoshi , Yamasaki Hideo
Published: October 2003

Peroxynitrite (ONOO−), a reactive nitrogen species, is capable of nitrating tyrosine residue of proteins. Here we show in vitro evidence that plant phenolic compounds can also be nitrated by an ONOO−‐independent mechanism. In the presence of NaNO2, H2O2, and horseradish peroxidase (HRP), monophenolic p‐coumaric acid (p‐CA, 4‐hydroxycinnamic acid) was nitrated to form 4‐hydroxy‐3‐nitrocinnamic acid. The reaction was completely inhibited by KCN, an inhibitor for HRP. The antioxidant ascorbate suppressed p‐CA nitration and its suppression time depended strongly on ascorbate concentration. We conclude that nitrogen dioxide radical (NO2

•
), but not ONOO−, produced by a guaiacol peroxidase is the intermediate for phytophenolic nitration.
Structural basis of ion pumping by Ca2+‐ATPase of sarcoplasmic reticulum
Authors: Toyoshima Chikashi , Nomura Hiromi , Sugita Yuji
Published: October 2003

The structures of the Ca2+‐ATPase (SERCA1a) have been determined for five different states by X‐ray crystallography. Detailed comparison of the structures in the Ca2+‐bound form and unbound (but thapsigargin‐bound) form reveals that very large rearrangements of the transmembrane helices take place accompanying Ca2+ dissociation and binding and that they are mechanically linked with equally large movements of the cytoplasmic domains. The meanings of the rearrangements of the transmembrane helices and those of the cytoplasmic domains, and the mechanistic roles of the phosphorylation are now becoming clear.
The lactose permease of Escherichia coli: overall structure, the sugar‐binding site and the alternating access model for transport
Authors: Abramson Jeff , Smirnova Irina , Kasho Vladimir , Verner Gillian , Iwata So , Kaback H.Ronald
Published: October 2003

Membrane transport proteins transduce free energy stored in electrochemical ion gradients into a concentration gradient and are a major class of membrane proteins, many of which play important roles in human health and disease. Recently, the X‐ray structure of the Escherichia coli lactose permease (LacY), an intensively studied member of a large group of related membrane transport proteins, was solved at 3.5 Å. LacY is composed of N‐ and C‐terminal domains, each with six transmembrane helices, symmetrically positioned within the molecule. The structure represents the inward‐facing conformation, as evidenced by a large internal hydrophilic cavity open to the cytoplasmic side. The structure with a bound lactose homolog reveals the sugar‐binding site in the cavity, and a mechanism for translocation across the membrane is proposed in which the sugar‐binding site has alternating accessibility to either side of the membrane.
Neurokinin 1‐receptors and sensory neuropeptides in tendon insertions at the medial and lateral epicondyles of the humerus Studies on tennis elbow and medial epicondylalgia
Authors: Ljung Björn‐Ove , Alfredson Håkan , Forsgren Sture
Published: October 2003

There is no information on the sensory innervation at the flexor muscle origin at the medial epicondyle of the humerus and it is not known if substance P receptors (Neurokinin 1‐receptors, NK1‐R) are present in tendon insertions in general. In the present investigation, we have studied the muscle origin in patients suffering from medial epicondylalgia and tennis elbow. Immunohistochemistry and antibodies to substance P (SP) and CGRP as well as the general nerve marker PGP 9.5 were used. Specific immunoreactions were observed in nerve bundles and as free nerve fibers. The immunoreactive structures were partly seen in association with some of the blood vessels. The observations constitute a morphological correlate for the occurrence of nerve mediated effects in this region. By using immunohistochemistry and antibodies to NK1‐R, the distribution of this receptor was studied at the insertion of the proximal tendon of the extensor carpi radialis brevis muscle at the lateral epicondyle. Specific immunoreactions were seen as varicose fibers occurring as single fibers or grouped into bundles, indicating that SP has effects in the nerves in this region. The results give further evidence for a possible neurogenic involvement in the pathophysiology of tennis elbow and in medial epicondylalgia. © 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.
Translation and Validation of the Breastfeeding Self‐Efficacy Scale Into Chinese
Authors: Dai Xiaona , Dennis Cindy‐Lee
Published: October 2003

The purpose of this methodological study was to translate the Breastfeeding Self‐Efficacy Scale (BSES) into Mandarin and determine the psychometric characteristics among a sample of 186 Chinese women. The BSES was translated and pilot‐tested with 21 breastfeeding women. Following minor revisions to ensure content and semantic equivalence, the instrument was administered to 186 hospitalized breastfeeding women, who were then contacted again at 4 and 8 weeks postpartum to determine their infant feeding method and readminister the Edinburgh Postnatal Depression Scale. The psychometric assessment method used to validate the original BSES (English version) was replicated with the translated Chinese version. The Cronbach's alpha coefficient for the Chinese version BSES was 0.93, suggesting good reliability. Consistent with the English version, the factor analysis procedures resulted in the theorized two‐factor solution and as hypothesized, the BSES was significantly correlated with the Edinburgh Postnatal Depression Scale, a theoretically related construct. Support for predictive validity was demonstrated through significant mean differences in BSES scores between breastfeeding and bottle‐feeding mothers at 4 and 8 weeks postpartum. The results from this study are the first to examine the psychometric characteristics of the Chinese version BSES administered in a hospital postnatally and provide further evidence of the reliability and validity of the instrument.
Arabidopsis Genes Encoding Mitochondrial Type II NAD(P)H Dehydrogenases Have Different Evolutionary Origin and Show Distinct Responses to Light
Authors: Michalecka Agnieszka M. , Svensson Å. Staffan , Johansson Fredrik I. , Agius Stephanie C. , Johanson Urban , Brennicke Axel , Binder Stefan , Rasmusson Allan G.
Published: October 2003

In addition to proton-pumping complex I, plant mitochondria contain several type II NAD(P)H dehydrogenases in the electron transport chain. The extra enzymes allow the nonenergy-conserving electron transfer from cytoplasmic and matrix NAD(P)H to ubiquinone. We have investigated the type II NAD(P)H dehydrogenase gene families in Arabidopsis. This model plant contains two and four genes closely related to potato (Solanum tuberosum) genes nda1 and ndb1, respectively. A novel homolog, termed ndc1, with a lower but significant similarity to potato nda1 and ndb1, is also present. All genes are expressed in several organs of the plant. Among the nda genes, expression of nda1, but not nda2, is dependent on light and circadian regulation, suggesting separate roles in photosynthesis-associated and other respiratory NADH oxidation. Genes from all three gene families encode proteins exclusively targeted to mitochondria, as revealed by expression of green fluorescent fusion proteins and by western blotting of fractionated cells. Phylogenetic analysis indicates that ndc1 affiliates with cyanobacterial type II NADH dehydrogenase genes, suggesting that this gene entered the eukaryotic cell via the chloroplast progenitor. The ndc1 should then have been transferred to the nucleus and acquired a signal for mitochondrial targeting of the protein product. Although they are of different origin, the nda, ndb, and ndc genes carry an identical intron position.
Down-Regulating α-Galactosidase Enhances Freezing Tolerance in Transgenic Petunia
Authors: Pennycooke Joyce C. , Jones Michelle L. , Stushnoff Cecil
Published: October 2003

α-Galactosidase (α-Gal; EC 3.2.1.22) is involved in many aspects of plant metabolism, including hydrolysis of the α-1,6 linkage of raffinose oligosaccharides during deacclimation. To examine the relationship between endogenous sugars and freezing stress, the expression of α-Gal was modified in transgenic petunia (Petunia × hybrida cv Mitchell). The tomato (Lycopersicon esculentum) Lea-Gal gene under the control of the Figwort Mosaic Virus promoter was introduced into petunia in the sense and antisense orientations using Agrobacterium tumefaciens-mediated transformation. RNA gel blots confirmed that α-Gal transcripts were reduced in antisense lines compared with wild type, whereas sense plants had increased accumulation of α-Gal mRNAs. α-Gal activity followed a similar trend, with reduced activity in antisense lines and increased activity in all sense lines evaluated. Raffinose content of nonacclimated antisense plants increased 12- to 22-fold compared with wild type, and 22- to 53-fold after cold acclimation. Based upon electrolyte leakage tests, freezing tolerance of the antisense lines increased from –4°C for cold-acclimated wild-type plants to –8°C for the most tolerant antisense line. Down-regulating α-Gal in petunia results in an increase in freezing tolerance at the whole-plant level in nonacclimated and cold-acclimated plants, whereas overexpression of the α-Gal gene caused a decrease in endogenous raffinose and impaired freezing tolerance. These results suggest that engineering raffinose metabolism by transformation with α-Gal provides an additional method for improving the freezing tolerance of plants.
wCLUTO: A Web-Enabled Clustering Toolkit
Authors: Rasmussen Matthew D. , Deshpande Mukund S. , Karypis George , Johnson James , Crow John A. , Retzel Ernest F.
Published: October 2003

As structural and functional genomics efforts provide the biological community with ever-broadening sets of interrelated data, the need to explore such complex information for subtle relationships expands. We present wCLUTO, a Web-enabled version of the stand-alone application CLUTO, designed to apply clustering methods to genomic information. Its first application is focused on the clustering transcriptome data from microarrays. Data can be uploaded by the user into the clustering tool, a choice of several clustering methods can be made and configured, and data are presented to the user in a variety of visual formats, including a three-dimensional “mountain” view of the clusters. Parameters can be explored to rapidly examine a variety of clustering results, and the resulting clusters can be downloaded either for manipulation by other programs or to be saved in a format for publication.
The Subcellular Localization of Plant Protein Phosphatase 5 Isoforms Is Determined by Alternative Splicing
Authors: de la Fuente van Bentem Sergio , Vossen Jack H. , Vermeer Josephus E.M. , de Vroomen Marianne J. , Gadella Theodorus W.J. , Haring Michel A. , Cornelissen Ben J.C.
Published: October 2003

Protein serine/threonine phosphatase 5 (PP5) plays an important role in signal transduction in animal cells, but in plants, knowledge about PP5 is scarce. Here, we describe the isolation of a full-length cDNA encoding tomato (Lycopersicon esculentum) PP5 (LePP5) and its expression in Escherichia coli. Biochemical characterization showed that recombinant LePP5 has a low intrinsic protein phosphatase activity. This activity was increased 6- to 10-fold by either removal of the N-terminal tetratricopeptide repeat domain or by addition of fatty acids, indicating that biochemical features specific for PP5 homologs from other species are conserved in tomato. The single-copy LePP5 gene was cloned and shown to encode two mRNA species that arise by alternative pre-mRNA splicing. Similarly, Arabidopsis was found to express two PP5 transcripts, suggesting that alternative splicing of PP5 pre-mRNA is not specific for tomato. Alternative splicing results in a larger transcript containing an additional exon encoding two putative transmembrane domains and, hence, in a larger PP5 isoform. Subcellular fractionation studies on tomato protein lysates indicated that the majority of the 55-kD LePP5 isoform is soluble, whereas the 62-kD isoform is an integral membrane protein. Production of yellow fluorescent protein-PP5 chimeras in plant cells indicated that the 55-kD isoform is localized in both the nucleus and the cytoplasm, whereas the 62-kD isoform is targeted to the endoplasmic reticulum, including the nuclear envelope. Our findings show that alternative splicing generates two LePP5 isoforms with a different subcellular localization.
The Polar Auxin Transport InhibitorN-1-Naphthylphthalamic Acid Disrupts Leaf Initiation, KNOX Protein Regulation, and Formation of Leaf Margins in Maize
Authors: Scanlon Michael J.
Published: October 2003

Maize (Zea mays) leaves develop basipetally (tip to base); the upper blade emerges from the shoot apical meristem (SAM) before the expansion of the lower sheath. Founder cells, leaf initials located in the periphery of the SAM, are distinguished from the SAM proper by the differential accumulation of KNOX proteins. KNOX proteins accumulate in the SAM, but are excluded from maize leaf primordia and leaf founder cells. As in Arabidopsis and tomato (Lycopersicon esculentum), maize shoots failed to initiate new leaves when cultured in the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). We demonstrate that NPA-induced arrest of leaf initiation in maize is correlated with the failure to down-regulate KNOX accumulation in the SAM. In addition, NPA-cultured shoots formed abnormal tubular leaf bases in which the margins failed to separate in the lower leaf zone. The tubular leaf bases always formed in the fourth leaf from the arrested meristem. Moreover, the unseparated margin domains of these tubular leaf bases accumulated ectopic KNOX protein(s). Transfer of NPA-cultured apices to NPA-free media resulted in the resumption of leaf initiation from the SAM and the restoration of normal patterns of KNOX down-regulation, accordingly. These data suggest that the lower sheath margins emerge from the leaf base late in maize leaf development and that the separation of these leaf margin domains is correlated with auxin transport and down-regulation of KNOX proteins. In addition, these results suggest that the down-regulation of KNOX accumulation in maize apices is not upstream of polar auxin transport, although a more complicated feedback network may exist. A model for L1-derived margin development in maize leaves is presented.
Mechanisms of Arsenical and Diamidine Uptake and Resistance in
Trypanosoma brucei
Authors: Matovu Enock , Stewart Mhairi L. , Geiser Federico , Brun Reto , Mäser Pascal , Wallace Lynsey J. M. , Burchmore Richard J. , Enyaru John C. K. , Barrett Michael P. , Kaminsky Ronald , Seebeck Thomas , de Koning Harry P.
Published: October 2003

Sleeping sickness, caused by
Trypanosoma brucei
spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In
T. brucei
, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene
TbAT1
has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of
TbAT1
in drug uptake and drug resistance in
T. brucei
by genetic knockout of
TbAT1. Tbat1
-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with
tbat1
-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.
The Road to Reconciliation: Antecedents of Victim Willingness to Reconcile Following a Broken Promise
Authors: Tomlinson Edward C. , Dineen Brian R. , Lewicki Roy J.
Published: October 2003

The antecedents of victim willingness to reconcile a professional relationship following an incident involving a broken promise were examined in terms of offender tactics (i.e., nature of apology, timeliness of reparative act, sincerity) and relationship characteristics (i.e., nature of past relationship, probability of future violation) using a within- and between-subjects policy-capturing design. Relatively speaking, relationship characteristics were as strongly related to willingness to reconcile as offender tactics. Furthermore, we found moderating effects of magnitude of violation on the willingness to reconcile a relationship following a trust violation. In particular, nature of past relationship was weighed more heavily, whereas probability of future violation was weighed less heavily when the magnitude of the violation was greater. Practical implications and recommendations for future research are discussed.
Rabbit knee model of post‐traumatic joint contractures: The long‐term natural history of motion loss and myofibroblasts
Authors: Hildebrand Kevin A. , Sutherland Craig , Zhang Mei
Published: October 2003

Our objective is to describe the natural history of motion loss with time and myofibroblast numbers in a rabbit knee model of post‐traumatic joint contractures. Twenty‐eight skeletally mature New Zealand White female rabbits had five‐mm‐squares of cortical bone removed from the medial and lateral femoral condyles of the right knee. A Kirschner wire (K‐wire) was used to immobilize the knee joint in maximum flexion. A second operation was performed 8 weeks later to remove the K‐wire. The rabbits were divided into four groups depending on the time of remobilization; 0, 8, 16 or 32 weeks.

The average flexion contracture of the experimental knees in the 0‐week and 8‐week remobilization groups (38° and 33°, respectively) were significantly greater when compared with the values of the unoperated contralateral knees (8°). The average flexion contractures of the experimental knees in the 16‐week and 32‐week remobilization groups were also greater than the unoperated contralateral knees, although they were not statistically significant. The average flexion contractures of the 16‐week and 32‐week groups were 19° and 18°, respectively, indicating a stabilization of the motion loss. Myofibroblast numbers in the posterior joint capsules were elevated 4–5×in the knees with contractures when compared to the contralateral knees. The initial decrease in severity followed by stabilization of motion loss and the association of motion loss with myofibroblasts mimics the human scenario of permanent post‐traumatic joint contractures. © 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.
Prediction of proteinase cleavage sites in polyproteins of coronaviruses and its applications in analyzing SARS‐CoV genomes
Authors: Gao Feng , Ou Hong-Yu , Chen Ling-Ling , Zheng Wen-Xin , Zhang Chun-Ting
Published: October 2003

Recently, we have developed a coronavirus‐specific gene‐finding system, ZCURVE_CoV 1.0. In this paper, the system is further improved by taking the prediction of cleavage sites of viral proteinases in polyproteins into account. The cleavage sites of the 3C‐like proteinase and papain‐like proteinase are highly conserved. Based on the method of traditional positional weight matrix trained by the peptides around cleavage sites, the present method also sufficiently considers the length conservation of non‐structural proteins cleaved by the 3C‐like proteinase and papain‐like proteinase to reduce the false positive prediction rate. The improved system, ZCURVE_CoV 2.0, has been run for each of the 24 completely sequenced coronavirus genomes in GenBank. Consequently, all the non‐structural proteins in the 24 genomes are accurately predicted. Compared with known annotations, the performance of the present method is satisfactory. The software ZCURVE_CoV 2.0 is freely available at http://tubic.tju.edu.cn/sars/.
Identification of previously undiagnosed left ventricular systolic dysfunction: community screening using natriuretic peptides and electrocardiography
Authors: Ng Leong L. , Loke Ian , Davies Joan E. , Khunti Kamlesh , Stone Margaret , Abrams Keith R. , Chin Derek T. , Squire Iain B.
Published: October 2003

Aims: We examined strategies to improve the positive predictive value of natriuretic peptides in screening for undiagnosed left ventricular systolic dysfunction (LVSD) in the community. Methods and results: The value of B‐type(BNP), N‐terminal proB‐type (N‐BNP) and N‐terminal proAtrial(N‐ANP) natriuretic peptides was prospectively assessed in 1360 subjects (45–80 years) together with echocardiography and electrocardiography. Seventeen individuals had definite and 13 had borderline, LVSD. Receiver‐operating‐characteristic (ROC) curve analysis showed the superiority of BNP (ROC areas 0.942 for definite LVSD, P<0.03; 0.934 for borderline LVSD, P<0.003) compared to N‐BNP or N‐ANP. Peptide levels, major ECG abnormality and ischaemic heart disease (IHD) history were independent predictors of LVSD. Logistic regression modelling incorporating these factors improved ROC areas for all natriuretic peptides. The specificity of all natriuretic peptides is enhanced by consideration of these factors. Conclusions: In population screening for definite LVSD, consideration of plasma natriuretic peptide levels together with the presence of major ECG abnormalities and IHD history reduces by a factor of six (in comparison to consideration of plasma natriuretic peptide levels in isolation) the number of subjects requiring echocardiography to detect one case of LVSD (for BNP, 44 falling to seven). Similar improvements were evident for N‐ANP and N‐BNP. Inclusion of major ECG abnormalities and IHD history improves the performance of any natriuretic peptide used in screening programmes for ruling in undiagnosed LVSD.