bluuebunny/crossref_metadata_2025 huggingface dataset:
Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human β-globin in engrafted mice
Authors: Kalberer Christian P. , Pawliuk Robert , Imren Suzan , Bachelot Thomas , Takekoshi Ken J. , Fabry Mary , Eaves Connie J. , London Irving M. , Humphries R. Keith , Leboulch Philippe
Published: July 2002

Transcriptional silencing of genes transferred into hematopoietic stem cells poses one of the most significant challenges to the success of gene therapy. If the transferred gene is not completely silenced, a progressive decline in gene expression as the mice age often is encountered. These phenomena were observed to various degrees in mouse transplant experiments using retroviral vectors containing a human β-globin gene, even when cis-linked to locus control region derivatives. Here, we have investigated whether
ex vivo
preselection of retrovirally transduced stem cells on the basis of expression of the green fluorescent protein driven by the CpG island phosphoglycerate kinase promoter can ensure subsequent long-term expression of a cis-linked β-globin gene in the erythroid lineage of transplanted mice. We observed that 100% of mice (
n
= 7) engrafted with preselected cells concurrently expressed human β-globin and the green fluorescent protein in 20–95% of their RBC for up to 9.5 mo posttransplantation, the longest time point assessed. This expression pattern was successfully transferred to secondary transplant recipients. In the presence of β-locus control region hypersensitive site 2 alone, human β-globin mRNA expression levels ranged from 0.15% to 20% with human β-globin chains detected by HPLC. Neither the proportion of positive blood cells nor the average expression levels declined with time in transplanted recipients. Although suboptimal expression levels and heterocellular position effects persisted,
in vivo
stem cell gene silencing and age-dependent extinction of expression were avoided. These findings support the further investigation of this type of vector for the gene therapy of human hemoglobinopathies.
Phosphorylation sites of protein kinase C δ in H
2
O
2
-treated cells and its activation by tyrosine kinase
in
vitro
Authors: Konishi Hiroaki , Yamauchi Emiko , Taniguchi Hisaaki , Yamamoto Toshiyoshi , Matsuzaki Hidenori , Takemura Yukitoshi , Ohmae Kyoko , Kikkawa Ushio , Nishizuka Yasutomi
Published: July 2002

Protein kinase C δ (PKC δ) is normally activated by
diacylglycerol produced from receptor-mediated
hydrolysis of inositol phospholipids. On stimulation of cells with
H
2
O
2
, the enzyme is tyrosine phosphorylated,
with a concomitant increase in enzymatic activity. This activation does
not appear to accompany its translocation to membranes. In the present
study, the tyrosine phosphorylation sites of PKC δ in the
H
2
O
2
-treated cells were identified as Tyr-311,
Tyr-332, and Tyr-512 by mass spectrometric analysis with the use of the
precursor-scan method and by immunoblot analysis with the use of
phosphorylation site-specific antibodies. Tyr-311 was the predominant
modification site among them. In an
in vitro
study,
phosphorylation at this site by Lck, a non-receptor-type tyrosine
kinase, enhanced the basal enzymatic activity and elevated its maximal
velocity in the presence of diacylglycerol. The
mutation of Tyr-311 to phenylalanine prevented the increase in this
maximal activity, but replacement of the other two tyrosine residues
did not block such an effect. The results indicate that phosphorylation
at Tyr-311 between the regulatory and catalytic domains is a critical
step for generation of the active PKC δ in response to
H
2
O
2
.
The effect of cultivation on the size, shape, and persistence of disease patches in fields
Authors: Truscott J. E. , Gilligan C. A.
Published: July 2002

Epidemics of soil-borne plant disease are characterized by patchiness because of restricted dispersal of inoculum. The density of inoculum within disease patches depends on a sequence comprising local amplification during the parasitic phase followed by dispersal of inoculum by cultivation during the intercrop period. The mechanisms that control size, shape, and persistence have received very little rigorous attention in epidemiological theory. Here we derive a model for dispersal of inoculum in soil by cultivation that takes account into the discrete stochastic nature of the system in time and space. Two parameters, probability of movement and mean dispersal distance, characterize lateral dispersal of inoculum by cultivation. The dispersal parameters are used in combination with the characteristic area and dimensions of host plants to identify criteria that control the shape and size of disease patches. We derive a critical value for the probability of movement for the formation of cross-shaped patches and show that this is independent of the amount of inoculum. We examine the interaction between local amplification of inoculum by parasitic activity and subsequent dilution by dispersal and identify criteria whereby asymptomatic patches may persist as inoculum falls below a threshold necessary for symptoms to appear in the subsequent crop. The model is motivated by the spread of rhizomania, an economically important soil-borne disease of sugar beet. However, the results have broad applicability to a very wide range of diseases that survive as discrete units of inoculum. The application of the model to patch dynamics of weed seeds and local introductions of genetically modified seeds is also discussed.
The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an
MLL
-fusion partner in acute myelogeneous leukemia
Authors: Fuchs Uta , Rehkamp Gönna , Haas Oskar A. , Slany Robert , König Margit , Bojesen Stig , Bohle Rainer M. , Damm-Welk Christine , Ludwig Wolf-Dieter , Harbott Jochen , Borkhardt Arndt
Published: July 2002

We have cloned a fusion partner of the
MLL
gene at 11q23 and identified it as the gene encoding the human formin-binding protein 17, FBP17. It maps to chromosome 9q34 centromeric to
ABL
. The gene fusion results from a complex chromosome rearrangement that was resolved by fluorescence
in situ
hybridization with various probes on chromosomes 9 and 11 as an ins(11;9)(q23;q34)inv(11)(q13q23). The rearrangement resulted in a 5′-
MLL
/
FBP17
-3′ fusion mRNA. We retrovirally transduced murine-myeloid progenitor cells with
MLL
/
FBP17
to test its transforming ability. In contrast
to MLL
/
ENL
,
MLL
/
ELL
and other
MLL
-fusion genes,
MLL
/
FBP17
did not give a positive readout in a serial replating assay. Therefore, we assume that additional cooperating genetic abnormalities might be needed to establish a full malignant phenotype. FBP17 consists of a C-terminal Src homology 3 domain and an N-terminal region that is homologous to the cell division cycle protein, cdc15, a regulator of the actin cytoskeleton in
Schizosaccharomyces pombe
. Both domains are separated by a consensus Rho-binding motif that has been identified in different Rho-interaction partners such as Rhotekin and Rhophilin. We evaluated whether FBP17 and members of the Rho family interact
in vivo
with a yeast two-hybrid assay. None of the various Rho proteins tested, however, interacted with FBP17. We screened a human kidney library and identified a sorting nexin, SNX2, as a protein interaction partner of FBP17. These data provide a link between the epidermal growth factor receptor pathway and an MLL fusion protein.
A negative feedback system between oocyte bone morphogenetic protein 15 and granulosa cell kit ligand: Its role in regulating granulosa cell mitosis
Authors: Otsuka Fumio , Shimasaki Shunichi
Published: July 2002

Although the existence of a regulatory paracrine feedback system between oocytes and follicular somatic cells has been postulated for some time, there has not yet been any definitive evidence that such a communication system exists. Herein we present a previously undescribed oocyte-granulosa cell (GC) feedback communication system involving an oocyte-derived factor, bone morphogenetic protein-15 (BMP-15) and a GC-derived factor, kit ligand (KL), both of which have been shown to be crucial regulators of female reproduction. We used a coculture system of rat oocytes and GCs and found that BMP-15 stimulates KL expression in GCs, whereas KL inhibits BMP-15 expression in oocytes, thus forming a negative feedback loop. Moreover, KL, like BMP-15, exhibited mitotic activity on GCs in the presence of oocytes. Because c-kit (KL receptor) is expressed in oocytes but not GCs, the oocytes must be involved in mediating the KL-induced GC mitosis. Furthermore, the blockage of c-kit signaling in oocytes by using a c-kit neutralizing antibody markedly suppressed BMP-15-induced GC mitosis, suggesting that the oocyte must play a role in the GC responses to BMP-15. In contrast, the c-kit antibody had no effect on the mitotic activities of two other known GC mitogens, activin-A and BMP-7. Altogether, this study presents direct evidence of a negative feedback system governed by oocyte-derived BMP-15 and GC-derived KL, and demonstrates that the mitotic activities of BMP-15 and KL for GCs depend on this oocyte–GC communication system. We hypothesize that the negative feedback system most likely plays a pivotal role in early folliculogenesis.
Timing of metamorphosis and the onset of the negative feedback loop between the thyroid gland and the pituitary is controlled by type II iodothyronine deiodinase in
Xenopus laevis
Authors: Huang Haochu , Cai Liquan , Remo Benjamin F. , Brown Donald D.
Published: July 2002

Two important features of amphibian metamorphosis are the sequential response of tissues to different concentrations of thyroid hormone (TH) and the development of the negative feedback loop between the pituitary and the thyroid gland that regulates TH synthesis by the thyroid gland. At the climax of metamorphosis in
Xenopus
laevis
(when the TH level is highest), the ratio of the circulating precursor thyroxine (T
4
) to the active form 3,5,3′-triiodothyronine (T
3
) in the blood is many times higher than it is in tissues. This difference is because of the conversion of T
4
to T
3
in target cells of the tadpole catalyzed by the enzyme type II iodothyronine deiodinase (D2) and the local effect (cell autonomy) of this activity. Limb buds and tails express D2 early and late in metamorphosis, respectively, correlating with the time that these organs undergo TH-induced change. T
3
is required to complete metamorphosis because the peak concentration of T
4
that is reached at metamorphic climax cannot induce the final morphological changes. At the climax of metamorphosis, D2 expression is activated specifically in the anterior pituitary cells that express the genes for thyroid-stimulating hormone but not in the cells that express proopiomelanocortin. Physiological concentrations of T
3
but not T
4
can suppress thyrotropin subunit β gene expression. The timing and the remarkable specificity of D2 expression in the thyrotrophs of the anterior pituitary coupled with the requirement for locally synthesized T
3
strongly support a role for D2 in the onset of the negative feedback loop at the climax of metamorphosis.
Nuclear particles containing RNA polymerase III complexes associated with the junctional plaque protein plakophilin 2
Authors: Mertens Claudia , Hofmann Ilse , Wang Zhengxin , Teichmann Martin , Chong Setareh Sepehri , Schnölzer Martina , Franke Werner W.
Published: July 2002

Plakophilin 2, a member of the arm-repeat protein family, is a dual location protein that occurs both in the cytoplasmic plaques of desmosomes as an architectural component and in an extractable form in the nucleoplasm. Here we report the existence of two nuclear particles containing plakophilin 2 and the largest subunit of RNA polymerase (pol) III (RPC155), both of which colocalize and are coimmunoselected with other pol III subunits and with the transcription factor TFIIIB. We also show that plakophilin 2 is present in the pol III holoenzyme, but not the core complex, and that it binds specifically to RPC155
in vitro
. We propose the existence of diverse nuclear particles in which proteins known as plaque proteins of intercellular junctions are complexed with specific nuclear proteins.
Energetic asymmetry among hydrogen bonds in MHC class II⋅peptide complexes
Authors: McFarland Benjamin J. , Katz John F. , Beeson Craig , Sant Andrea J.
Published: July 2002

Comparison of crystallized MHC class II⋅peptide complexes has revealed that, in addition to pocket interactions involving the peptide side chains, peptide binding to MHC class II molecules is characterized by a series of hydrogen bonds between genetically conserved amino acid residues in the class II molecule and the main chain of the peptide. Many class II⋅peptide structures have two sets of symmetrical hydrogen bonds at the opposite ends of the class II antigen-binding groove (β-His-81, β-Asn-82 vs. α-His-68, α-Asn-69). In this study, we alter these peripheral hydrogen bonds and measure the apparent contribution of each to the kinetic stability of peptide⋅class II complexes. Single conservative amino substitutions were made in the I-Adprotein to eliminate participation as a hydrogen bonding residue, and the kinetic stability of a diverse set of peptides bound to the substituted I-Adproteins was measured. Although each hydrogen bond does contribute to peptide binding, our results point to the striking conclusion that those hydrogen bonds localized to the amino terminus of the peptide contribute profoundly and disproportionately to the stability of peptide interactions with I-Ad. We suggest that the peripheral hydrogen bonds at the amino terminus of the bound peptide that are conserved in all class II⋅peptide crystal structures solved thus far form a cooperative network that critically regulates peptide dissociation from the class II molecule.
Engineering blood meal-activated systemic immunity in the yellow fever mosquito,
Aedes aegypti
Authors: Kokoza Vladimir , Ahmed Abduelaziz , Cho Wen-Long , Jasinskiene Nijole , James Anthony A. , Raikhel Alexander
Published: July 2002

Progress in molecular genetics makes possible the development of alternative disease control strategies that target the competence of mosquitoes to transmit pathogens. We tested the regulatory region of the
vitellogenin
(
Vg
) gene of
Aedes aegypti
for its ability to express potential antipathogen factors in transgenic mosquitoes.
Hermes
-mediated transformation was used to integrate a 2.1-kb
Vg
-promoter fragment driving the expression of the
Defensin A
(
DefA
) coding region, one of the major insect immune factors. PCR amplification of genomic DNA and Southern blot analyses, carried out through the ninth generation, showed that the
Vg-DefA
transgene insertion was stable. The
Vg-DefA
transgene was strongly activated in the fat body by a blood meal. The mRNA levels reached a maximum at 24-h postblood meal, corresponding to the peak expression time of the endogenous
Vg
gene. High levels of transgenic defensin were accumulated in the hemolymph of bloodfed female mosquitoes, persisting for 20–22 days after a single blood feeding. Purified transgenic defensin showed antibacterial activity comparable to that of defensin isolated from bacterially challenged control mosquitoes. Thus, we have been able to engineer the genetically stable transgenic mosquito with an element of systemic immunity, which is activated through the blood meal-triggered cascade rather than by infection. This work represents a significant step toward the development of molecular genetic approaches to the control of vector competence in pathogen transmission.
Thermodynamics of the interplay between magnetism and high-temperature superconductivity
Authors: Kivelson Steven A. , Aeppli G. , Emery Victor J.
Published: July 2002

Copper–oxide-based high-temperature superconductors have complex phase diagrams with multiple ordered phases. It even appears that the highest superconducting transition temperatures for certain cuprates are found in samples that display simultaneous onset of magnetism and superconductivity. We show here how the thermodynamics of fluid mixtures—a touchstone for chemistry as well as hard and soft condensed matter physics—accounts for this startling observation, as well as many other properties of the cuprates in the vicinity of the instability toward “striped” magnetism.
Specific treatment of autoimmunity with recombinant invariant chains in which CLIP is replaced by self-epitopes
Authors: Bischof Felix , Wienhold Wolfgang , Wirblich Christoph , Malcherek Georg , Zevering Olayinka , Kruisbeek Ada M. , Melms Arthur
Published: July 2002

The invariant chain (Ii) binds to newly synthesized MHC class II molecules with the CLIP region of Ii occupying the peptide-binding groove. Here we demonstrate that recombinant Ii proteins with the CLIP region replaced by antigenic self-epitopes are highly efficient in activating and silencing specific T cellsin vitroandin vivo. The Ii proteins require endogenous processing by antigen-presenting cells for efficient T cell activation. An Ii protein encompassing the epitope myelin basic protein amino acids 84–96 (Ii-MBP84–96) induced the model autoimmune disease experimental allergic encephalomyelitis (EAE) with a higher severity and earlier onset than the peptide. When applied in a tolerogenic manner, Ii-MBP84–96 abolished antigen-specific T cell proliferation and suppressed peptide-induced EAE more effectively than peptide alone. Importantly, i.v. administration of Ii proteins after EAE induction completely abrogated the disease, whereas peptides only marginally suppressed disease symptoms. Ii fusion proteins are thus more efficient than peptide in modulating CD4+T cell-mediated autoimmunity, documenting their superior qualities for therapeutic antigen deliveryin vivo.
Glucose depletion causes haploid invasive growth in yeast
Authors: Cullen Paul J. , Sprague George F.
Published: July 2002

Haploid yeast invades solid agar in response to nutrient
limitation. To decipher the cues that underlie invasion, we have
developed a single cell invasive growth assay. Using this assay, as
well as the traditional plate-washing assay, we show that invasive
growth occurs in response to glucose depletion. In the absence of
glucose (or other fermentable sugar), individual cells adopted a
nonaxial budding pattern and elongated morphology within the first cell
divisions, and invasion into the agar was observed in microcolonies
containing as few as 10 cells. In support of this observation, we found
that glucose suppressed the hyperinvasive growth morphology of
STE11
-
4, pbs2, hsl7
, and
RAS2V19
mutations. In addition, removal of glucose from
YPD medium caused constitutive invasion in wild-type cells. We tested
glucose control proteins for a role in invasion and found that Snf1, a
protein required for derepression of glucose-repressed genes, was
required for invasive growth. The transcription factor Sip4, which
interacts with Snf1 and is induced during the diauxic shift, had an
inhibitory role on invasive growth, suggesting that multiple mechanisms
are required for glucose depletion-dependent invasion.
A cyclin-dependent kinase-activating kinase regulates differentiation of root initial cells in
Arabidopsis
Authors: Umeda Masaaki , Umeda-Hara Chikage , Uchimiya Hirofumi
Published: July 2002

Cell division and differentiation continue throughout the plant
life cycle without significant loss of control. However, little is
known about the mechanisms that allow the continuous development of
meristems. Cell division is controlled by a family of cyclin-dependent
kinases (CDKs). CDK-activating kinases (CAKs) are known to
phosphorylate and activate almost all CDKs and thus may have a crucial
role in controlling CDK activities in each cell of the meristems. Here,
we show that overexpression of sense or antisense gene for Cak1At in
Arabidopsis
by using the glucocorticoid-mediated
transcriptional induction system resulted in a reduction of CDK
activities. After 14–24 h of glucocorticoid treatment, starch granules
appeared in columellar initials in the root meristem, and cortical
initials were periclinally divided into cortical and endodermal cells.
Accumulation of the cyclin∷β-glucuronidase fusion protein ceased
after 72 h of glucocorticoid treatment. Our results indicate that
a change of Cak1At activity leads to differentiation of initial cells,
followed by cessation of cell division. Therefore, we propose that
differentiation of initial cells is controlled by Cak1At but is
maintained independent of cell division.
Ama1p is a meiosis-specific regulator of the anaphase promoting complex/cyclosome in yeast
Authors: Cooper Katrina F. , Mallory Michael J. , Egeland Daniel B. , Jarnik Michal , Strich Randy
Published: July 2002

Meiosis is the developmental program by which diploid
organisms produce haploid gametes capable of sexual reproduction. Here
we describe the yeast gene
AMA1
, a new member of the
Cdc20 protein family that regulates the multisubunit ubiquitin ligase
termed the anaphase promoting complex/cyclosome (APC/C).
AMA1
is developmentally regulated in that its
transcription and splicing occur only in meiotic cells. The
meiosis-specific processing of
AMA1
mRNA depends on the
previously described
MER1
splicing factor. Several
results indicate that Ama1p is required for APC/C function during
meiosis. First, coimmunoprecipitation assays indicate that Ama1p
associates with the APC/C
in vivo
. Second, Ama1p is
required for the degradation of the B-type cyclin Clb1p, an APC/C
substrate in both meiotic and mitotic cells. Third, ectopic
overexpression of
AMA1
is able to stimulate
ubiquitination of Clb1p
in vitro
and degradation of
Clb1p
in vivo
. Mutants lacking
AMA1
revealed that it is required for the first meiotic division but not the
mitotic-like meiosis II. In addition,
ama1
mutants are
defective for both spore wall assembly and the expression of late
meiotic genes. In conclusion, this study indicates that Ama1p directs a
meiotic APC/C that functions solely outside mitotic cell division.
The requirement of Ama1p only for meiosis I and spore
morphogenesis suggests a function for APC/C
Ama1
specifically adapted to germ cell development.
Protein-protein interactions in the rigor actomyosin complex.
Authors: Milligan R A
Published: July 2002

Since it has not been possible to crystallize the actomyosin complex, the x-ray structures of the individual proteins together with data obtained by fiber diffraction and electron microscopy have been used to build detailed models of filamentous actin (f-actin) and the actomyosin rigor complex. In the f-actin model, a single monomer uses 10 surface loops and two alpha-helices to make sometimes complicated interactions with its four neighbors. In the myosin molecule, both the essential and regulatory light chains show considerable structural homology to calmodulin. General principles are evident in their mode of attachment to the target alpha-helix of the myosin heavy chain. The essential light chain also makes contacts with other parts of the heavy chain and with the regulatory light chain. The actomyosin rigor interface is extensive, involving interaction of a single myosin head with regions on two adjacent actin monomers. A number of hydrophobic residues on the apposing faces of actin and myosin contribute to the main binding site. This site is flanked on three sides by charged myosin surface loops that form predominantly ionic interactions with adjacent regions of actin. Hydrogen bonding is likely to play a significant role in actin-actin and actin-myosin interactions since many of the contacts involve loops. The model building approach used with actomyosin is applicable to other multicomponent assemblies of biological interest and is a powerful method for revealing molecular interactions and providing insights into the mode of action of the assemblies.
Direct observation of fast protein folding: the initial collapse of apomyoglobin.
Authors: Ballew R M , Sabelko J , Gruebele M
Published: July 2002

The rapid refolding dynamics of apomyoglobin are followed by a new temperature-jump fluorescence technique on a 15-ns to 0.5-ms time scale in vitro. The apparatus measures the protein-folding history in a single sweep in standard aqueous buffers. The earliest steps during folding to a compact state are observed and are complete in under 20 micros. Experiments on mutants and consideration of steady-state CD and fluorescence spectra indicate that the observed microsecond phase monitors assembly of an A x (H x G) helix subunit. Measurements at different viscosities indicate diffusive behavior even at low viscosities, in agreement with motions of a solvent-exposed protein during the initial collapse.
Gadolinium(III) texaphyrin: a tumor selective radiation sensitizer that is detectable by MRI.
Authors: Young S W , Qing F , Harriman A , Sessler J L , Dow W C , Mody T D , Hemmi G W , Hao Y , Miller R A
Published: July 2002

Gadolinium(III) texaphyrin (Gd-tex2+) is representative of a new class of radiation sensitizers detectable by magnetic resonance imaging (MRI). This porphyrin-like complex has a high electron affinity [E1/2 (red.) approximately = -0.08 V versus normal hydrogen electrode] and forms a long-lived pi-radical cation upon exposure to hydrated electrons, reducing ketyl radicals, or superoxide ions. Consistent with these chemical findings, Gd-tex2+ was found to be an efficient radiation sensitizer in studies carried out with HT29 cells in in vitro as well as in in vivo single and multifraction irradiation studies with a murine mammary carcinoma model. Selective localization of Gd-tex2+ in tumors was confirmed by MRI scanning.
A change of ploidy can modify epigenetic silencing.
Authors: Mittelsten Scheid O , Jakovleva L , Afsar K , Maluszynska J , Paszkowski J
Published: July 2002

A silent transgene in Arabidopsis thaliana was reactivated in an outcross but not upon selfing of hemizygous plants. This result could only be explained by assuming a genetic difference between the transgene-free gametes of the wild-type and hemizygous transgenic plants, respectively, and led to the discovery of ploidy differences between the parental plants. To investigate whether a change of ploidy by itself can indeed influence gene expression, we performed crosses of diploid or tetraploid plants with a strain containing a single copy of a transgenic resistance gene in an active state. We observed reduced gene expression of the transgene in triploid compared with diploid hybrids. This led to loss of the resistant phenotype at various stages of seedling development in part of the population. The gene inactivation was reversible. Thus, an increased number of chromosomes can result in a new type of epigenetic gene inactivation, creating differences in gene expression patterns. We discuss the possible impact of this finding for genetic diploidization in the light of widespread, naturally occurring polyploidy and polysomaty in plants.
Multiple forms of complement C3 in trout that differ in binding to complement activators.
Authors: Sunyer J O , Zarkadis I K , Sahu A , Lambris J D
Published: July 2002

In all other species analyzed to date, the functionally active form of complement component C3 exists as the product of a single gene. We have now identified and characterized three functional C3 proteins (C3-1, C3-3, and C3-4) in trout that are the products of at least two distinct C3 genes. All three proteins are composed of an alpha-and a beta-chain and contain a thioester bond in the alpha-chain. However, they differ in their electrophoretic mobility, glycosylation, reactivity with monospecific C3 antibodies, and relative ability to bind to various surfaces (zymosan, Escherichia coli, erythrocytes). A comparison of the partial amino acid sequences of the three proteins showed that the amino acid sequence identity/similarity of C3-3 to C3-4 is 87/91%, while that of C3-3 and C3-4 to C3-1 is 51.5/65.5% and 60/73% respectively. Thus, trout possess multiple forms of functional C3 that represent the products of several distinct genes and differ in their ability to bind covalently to various complement activators.
16 alpha-substituted analogs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity.
Authors: Wagner B L , Pollio G , Leonhardt S , Wani M C , Lee D Y , Imhof M O , Edwards D P , Cook C E , McDonnell D P
Published: July 2002

Previously, we have shown that agonists and antagonists interact with distinct, though overlapping regions within the human progesterone receptor (hPR) resulting in the formation of structurally different complexes. Thus, a link was established between the structure of a ligand-receptor complex and biological activity. In this study, we have utilized a series of in vitro assays with which to study hPR pharmacology and have identified a third class of hPR ligands that induce a receptor conformation which is distinct from that induced by agonists or antagonists. Importantly, when assayed on PR-responsive target genes these compounds were shown to exhibit partial agonist activity; an activity that was influenced by cell context. Thus, as has been shown previously for estrogen receptor, the overall structure of the ligand-receptor complex is influenced by the nature of the ligand. It appears, therefore, that the observed differences in the activity of some PR and estrogen receptor ligands reflect the ability of the cellular transcription machinery to discriminate between the structurally different complexes that result following ligand interaction. These data support the increasingly favored hypothesis that different ligands can interact with different regions within the hormone binding domains of steroid hormone receptors resulting in different biologies.
Lipid thioesters derived from acylated proteins accumulate in infantile neuronal ceroid lipofuscinosis: correction of the defect in lymphoblasts by recombinant palmitoyl-protein thioesterase.
Authors: Lu J Y , Verkruyse L A , Hofmann S L
Published: July 2002

Palmitoyl-protein thioesterase is a lysosomal long-chain fatty acyl hydrolase that removes fatty acyl groups from modified cysteine residues in proteins. Mutations in palmitoyl-protein thioesterase were recently found to cause the neurodegenerative disorder infantile neuronal ceroid lipofuscinosis, a disease characterized by accumulation of amorphous granular deposits in cortical neurons, leading to blindness, seizures, and brain death by the age of three. In the current study, we demonstrate that [35S]cysteine-labeled lipid thioesters accumulate in immortalized lymphoblasts of patients with infantile neuronal ceroid lipofuscinosis. The accumulation in cultured cells is reversed by the addition of recombinant palmitoyl-protein thioesterase that is competent for lysosomal uptake through the mannose-6-phosphate receptor. The [35S]cysteine-labeled lipids are substrates for palmitoyl-protein thioesterase in vitro, and their formation requires prior protein synthesis. These data support a role for palmitoyl-protein thioesterase in the lysosomal degradation of S-acylated proteins and define a major new pathway for the catabolism of acylated proteins in the lysosome.
Plant virus DNA replication processes in Agrobacterium: insight into the origins of geminiviruses?
Authors: Rigden J E , Dry I B , Krake L R , Rezaian M A
Published: July 2002

Agrobacterium tumefaciens, a bacterial plant pathogen, when transformed with plasmid constructs containing greater than unit length DNA of tomato leaf curl geminivirus accumulates viral replicative form DNAs indistinguishable from those produced in infected plants. The accumulation of the viral DNA species depends on the presence of two origins of replication in the DNA constructs and is drastically reduced by introducing mutations into the viral replication-associated protein (Rep or C1) ORF, indicating that an active viral replication process is occurring in the bacterial cell. The accumulation of these viral DNA species is not affected by mutations or deletions in the other viral open reading frames. The observation that geminivirus DNA replication functions are supported by the bacterial cellular machinery provides evidence for the theory that these circular single-stranded DNA viruses have evolved from prokaryotic episomal replicons.
Single residue substitutions that change the gating properties of a mechanosensitive channel in Escherichia coli.
Authors: Blount P , Sukharev S I , Schroeder M J , Nagle S K , Kung C
Published: July 2002

MscL is a channel that opens a large pore in the Escherichia coli cytoplasmic membrane in response to mechanical stress. Previously, we highly enriched the MscL protein by using patch clamp as a functional assay and cloned the corresponding gene. The predicted protein contains a largely hydrophobic core spanning two-thirds of the molecule and a more hydrophilic carboxyl terminal tail. Because MscL had no homology to characterized proteins, it was impossible to predict functional regions of the protein by simple inspection. Here, by mutagenesis, we have searched for functionally important regions of this molecule. We show that a short deletion from the amino terminus (3 amino acids), and a larger deletion of 27 amino acids from the carboxyl terminus of this protein, had little if any effect in channel properties. We have thus narrowed the search of the core mechanosensitive mechanism to 106 residues of this 136-amino acid protein. In contrast, single residue substitutions of a lysine in the putative first transmembrane domain or a glutamine in the periplasmic loop caused pronounced shifts in the mechano-sensitivity curves and/or large changes in the kinetics of channel gating, suggesting that the conformational structure in these regions is critical for normal mechanosensitive channel gating.
Redesigning secondary structure to invert coenzyme specificity in isopropylmalate dehydrogenase.
Authors: Chen R , Greer A , Dean A M
Published: July 2002

Rational engineering of enzymes involves introducing key amino acids guided by a knowledge of protein structure to effect a desirable change in function. To date, all successful attempts to change specificity have been limited to substituting individual amino acids within a protein fold. However, the infant field of protein engineering will only reach maturity when changes in function can be generated by rationally engineering secondary structures. Guided by x-ray crystal structures and molecular modeling, site-directed mutagenesis has been used to systematically invert the coenzyme specificity of Thermus thermophilus isopropylmalate dehydrogenase from a 100-fold preference for NAD to a 1000-fold preference for NADP. The engineered mutant, which is twice as active as wild type, contains four amino acid substitutions and an alpha-helix and loop that replaces the original beta-turn. These results demonstrate that rational engineering of secondary structures to produce enzymes with novel properties is feasible.
A tumor-selective somatostatin analog (TT-232) with strong in vitro and in vivo antitumor activity.
Authors: Kéri G , Erchegyi J , Horváth A , Mezõ I , Idei M , Vántus T , Balogh A , Vadász Z , Bökönyi G , Seprõdi J , Teplán I , Csuka O , Tejeda M , Gaál D , Szegedi Z , Szende B , Roze C , Kalthoff H , Ullrich A
Published: July 2002

We report a series of new in vitro and in vivo data proving the selective antitumor activity of our somatostatin structural derivative, TT-232. In vitro, it inhibited the proliferation of 20 different human tumor cell lines in the range of 50-95% and induced a very strong apoptosis. In vivo TT-232 was effective on transplanted animal tumors (Colon 26, B16 melanoma, and S180 sarcoma) and on human tumor xenografts. Treatment of MDA-MB-231 human breast cancer xenografted in mice with low submaximal doses of TT-232 [0.25 and 0.5 mg/kg of body weight (b.w.)] caused an average 80% decrease in the tumor volume resulting in 30% tumor-free animals surviving for longer than 200 days. Treatment of prostate tumor (PC-3) xenografted animals with 20 mg/kg of b.w. of TT-232 for 3 weeks resulted in 60% decrease in tumor volume and 100% survival even after 60 days, while 80% of nontreated animals perished. We have demonstrated that TT-232 did not bind to the membrane preparation of rat pituitary and cortex and had no antisecretory activity. TT-232 was not toxic at a dose of 120 mg/kg of b.w. in mice. Long-term incubation (24 h) of tumor cells with TT-232 caused significant inhibition of tyrosine kinases in good correlation with the apoptosis-inducing effect. The level of p53 or KU86 did not change following TT-232 treatment, suggesting a p53-independent apoptotic effect. Preincubation of human breast cancer cells (MDA-MB-453) with TT-232 for 2 h decreased the growth factor receptor autophosphorylation. All of these data suggest that TT-232 is a promising and selective antitumor agent.
Defective subunit assembly underlies a digenic form of retinitis pigmentosa linked to mutations in peripherin/rds and rom-1
Authors: Goldberg Andrew F. X. , Molday Robert S.
Published: July 2002

Retinitis pigmentosa (RP) is a group of progressive retinal
dystrophies that include the most common hereditary degenerative
diseases affecting the retina. Although most disease phenotypes appear
to result from defects at single genetic loci (monogenic), at least one
instance of RP appears to require a coinheritance of defects in the
unlinked peripherin/rds and rom-1 alleles (digenic), which encode the
polypeptide subunits of an oligomeric transmembrane protein complex
present at photoreceptor outer segment disc rims. Sedimentation
velocity analysis was performed upon the affected gene products
expressed heterologously in COS-1 cells to examine the assembly of the
subunit polypeptides. The results indicate that the missense
peripherin/rds mutant, L185P, which segregates with instances of
digenically inherited RP, is conditionally defective with respect to
its subunit assembly. Unlike wild-type peripherin/rds, the L185P
mutant does not form native-like homotetramers on its own; however, the
L185P mutant can assemble with wild-type rom-1 to form a structurally
normal heterotetrameric complex. These findings provide a novel
molecular-based rationale for the unusual digenic disease inheritance
pattern and offer insight into regions of peripherin/rds and rom-1,
which contribute to subunit–subunit interactions.
Heat shock induces a loss of rRNA-encoding DNA repeats in Brassica nigra.
Authors: Waters E R , Schaal B A
Published: July 2002

Stress-induced mutations may play an important role in the evolution of plants. Plants do not sequester a germ line, and thus any stress-induced mutations could be passed on to future generations. We report a study of the effects of heat shock on genomic components of Brassica nigra Brassicaceae. Plants were submitted to heat stress, and the copy number of two nuclear-encoded single-copy genes, rRNA-encoding DNA (rDNA) and a chloroplast DNA gene, was determined and compared to a nonstressed control group. We determined whether genomic changes were inherited by examining copy number in the selfed progeny of control and heat-treated individuals. No effects of heat shock on copy number of the single-copy nuclear genes or on chloroplast DNA are found. However, heat shock did cause a statistically significant reduction in rDNA copies inherited by the F1 generation. In addition, we propose a DNA damage-reppair hypothesis to explain the reduction in rDNA caused by heat shock.
Direct measurement of salt-bridge solvation energies using a peptide model system: implications for protein stability.
Authors: Wimley W C , Gawrisch K , Creamer T P , White S H
Published: July 2002

The solvation energies of salt bridges formed between the terminal carboxyl of the host pentapeptide AcWL- X-LL and the side chains of Arg or Lys in the guest (X) position have been measured. The energies were derived from octanol-to-buffer transfer free energies determined between pH 1 and pH 9. 13C NMR measurements show that the salt bridges form in the octanol phase, but not in the buffer phase, when the side chains and the terminal carboxyl group are charged. The free energy of salt-bridge formation in octanol is approximately -4 kcal/mol (1 cal = 4.184 J), which is equal to or slightly larger than the sum of the solvation energies of noninteracting pairs of charged side chains. This is about one-half the free energy that would result from replacing a charge pair in octanol with a pair of hydrophobic residues of moderate size. Therefore, salt bridging in octanol can change the favorable aqueous solvation energy of a pair of oppositely charged residues to neutral or slightly unfavorable but cannot provide the same free energy decrease as hydrophobic residues. This is consistent with recent computational and experimental studies of protein stability.
Ethinylestradiol does not enhance the expression of nitric oxide synthase in bovine endothelial cells but increases the release of bioactive nitric oxide by inhibiting superoxide anion production.
Authors: Arnal J F , Clamens S , Pechet C , Negre-Salvayre A , Allera C , Girolami J P , Salvayre R , Bayard F
Published: July 2002

Estradiol is known to exert a protective effect against the development of atherosclerosis, but the mechanism by which this protection is mediated is unclear. Since animal studies strongly suggest that production of endothelium-derived relaxing factor is enhanced by estradiol, we have examined the effect of estrogens on nitric oxide (NO) synthase (NOS) activity, protein, and mRNA in cultured bovine aortic endothelial cells. In reporter cells rich in guanylate cyclase, it has been observed that long-term treatment (> or = 24 hr) with ethinylestradiol (EE2) dose-dependently increased guanylate cyclase-activating factor activity in the conditioned medium of endothelial cells. However, conversion of L-[14C]arginine to L-[14C]citrulline by endothelial cell homogenate or quantification of nitrite and nitrate released by intact cells in the conditioned medium did not reveal any change in NOS activity induced by EE2 treatment. Similarly, Western and Northern blot analyses did not reveal any change in the endothelial NOS protein and mRNA content in response to EE2. However, EE2 dose- and time-dependently decreased superoxide anion production in the conditioned medium of endothelial cells with an EC50 value (0.1 nM) close to that which increased guanylate cyclase-activating factor activity (0.5 nM). Both of these effects were completely prevented by the antiestrogens tamoxifen and RU54876. Thus, endothelium exposure to estrogens appears to induce a receptor-mediated antioxidant effect that enhances the biological activity of endothelium-derived NO. These effects could account at least in part for the vascular protective properties of these hormones.
Surface dynamics in living acinar cells imaged by atomic force microscopy: Identification of plasma membrane structures involved in exocytosis
Authors: Schneider Stefan W. , Sritharan Kumudesh C. , Geibel John P. , Oberleithner Hans , Jena Bhanu P.
Published: July 2002

The dynamics at the plasma membrane resulting from secretory
vesicle docking and fusion and compensatory endocytosis has been
difficult to observe in living cells primarily due to limited
resolution at the light microscopic level. Using the atomic force
microscope, we have been able to image and record changes in plasma
membrane structure at ultrahigh resolution after stimulation of
secretion from isolated pancreatic acinar cells. “Pits” measuring
500–2000 nm and containing 3–20 depressions measuring 100–180 nm in
diameter were observed only at the apical region of acinar cells. The
time course of an increase and decrease in “depression” size
correlated with an increase and decrease of amylase secretion from live
acinar cells. Depression dynamics and amylase release were found to be
regulated in part by actin. No structural changes were identified at
the basolateral region of these cells. Our results suggest depressions
to be the fusion pores identified earlier in mast cells by
freeze-fracture electron microscopy and by electrophysiological
measurements. The atomic force microscope has enabled us to observe
plasma membrane dynamics of the exocytic process in living cells in
real time.
RNA polymerase sigma factor determines start-site selection but is not required for upstream promoter element activation on heteroduplex (bubble) templates
Authors: Fredrick Kurt , Helmann John D.
Published: July 2002

Sequence-selective transcription by bacterial RNA polymerase (RNAP) requires σ factor that participates in both promoter recognition and DNA melting. RNAP lacking σ (core enzyme) will initiate RNA synthesis from duplex ends, nicks, gaps, and single-stranded regions. We have used DNA templates containing short regions of heteroduplex (bubbles) to compare initiation in the presence and absence of various σ factors. Using bubble templates containing the σ
D
-dependent flagellin promoter, with or without its associated upstream promoter (UP) element, we demonstrate that UP element stimulation occurs efficiently even in the absence of σ. This supports a model in which the UP element acts primarily through the α subunit of core enzyme to increase the initial association of RNAP with the promoter. Core and holoenzyme do differ substantially in the template positions chosen for initiation: σ
D
restricts initiation to sites 8–9 nucleotides downstream of the conserved −10 element. Remarkably, σ
A
also has a dramatic effect on start-site selection even though the σ
A
holoenzyme is inactive on the corresponding homoduplexes. The start sites chosen by the σ
A
holoenzyme are located 8 nucleotides downstream of sequences on the nontemplate strand that resemble the conserved −10 hexamer recognized by σ
A
. Thus, σ
A
appears to recognize the −10 region even in a single-stranded state. We propose that in addition to its described roles in promoter recognition and start-site melting, σ also localizes the transcription start site.
Resolution of an early RecA-recombination intermediate by a junction-specific endonuclease
Authors: Chiu Sung Kay , Low K. Brooks , Yuan Andrew , Radding Charles M.
Published: July 2002

The nucleoprotein filament formed on a circular single strand by
Escherichia coli
RecA protein
in vitro
can pair with homologous duplex DNA even when the latter lacks a free homologous end, but subsequent progression of the reaction through strand exchange requires an end in at least one strand of the duplex DNA. We purified from
E. coli
an endonuclease activity that cleaves the outgoing strand of duplex DNA at the junction of homologous and heterologous sequences in three-stranded RecA-recombination intermediates. This endonuclease activity also cleaves specifically at the junctions of duplex and single-stranded regions in synthetic double-stranded oligonucleotides whose central portion consists of unpaired heterologous sequences. These activities are consistent with a role in recombination and repair of DNA.