bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Bcl-xLexpression in human keratinocytes by cell–substratum adhesion and the epidermal growth factor receptor
Authors: Rodeck Ulrich , Jost Monika , DuHadaway James , Kari Csaba , Jensen Pamela J. , Risse Barbara , Ewert Donald L.
Published: July 2002

Cell–substratum adhesion is an essential requirement for survival of human neonatal keratinocytesin vitro. Similarly, activation of the epidermal growth factor receptor (EGF-R) has recently been implicated not only in cell cycle progression but also in survival of normal keratinocytes. The mechanisms by which either cell–substratum adhesion or EGF-R activation protect keratinocytes from programmed cell death are poorly understood. Here we describe that blockade of the EGF-R and inhibition of substratum adhesion share a common downstream event, the down-regulation of the cell death protector Bcl-xL. Expression of Bcl-xLprotein was down-regulated during forced suspension culture of keratinocytes, concurrent with large-scale apoptosis. Similarly, EGF-R blockade was accompanied by down-regulation of Bcl-xLsteady-state mRNA and protein levels to an extent comparable to that observed in forced suspension culture. However, down-regulation of Bcl-xLexpression by EGF-R blockade was not accompanied by apoptosis; in this case, a second signal, generated by passaging, was required to induce rapid and large-scale apoptosis. These findings are consistent with the conclusions that (i) Bcl-xLrepresents a shared molecular target for signaling through cell-substrate adhesion receptors and the EGF-R, and (ii) reduced levels of Bcl-xLexpression through EGF-R blockade lower the tolerance of keratinocytes for cell death signals generated by cellular stress.
Diffusion control in an elementary protein folding reaction
Authors: Jacob Maik , Schindler Thomas , Balbach Jochen , Schmid Franz X.
Published: July 2002

The cold-shock protein CspB (from
Bacillus
subtilis
), a very small protein of 67 residues, folds extremely
fast in a reversible N ⇋ U two-state reaction. Both unfolding and
refolding are strongly decelerated when the viscosity of the solvent is
increased by adding ethylene glycol or sucrose. The folding of CspB
thus seems to follow Kramers’ model for reactions in which the
reactants must diffuse together. It indicates that the compaction of
the protein chain occurs in the rate-limiting step of folding. Chain
diffusion to a productively collapsed form and the crossing of a high
energy barrier are thus tightly coupled in this folding reaction, and
the measured reaction rate depends on both the diffusion of the protein
chain in the solvent and the magnitude of the activation energy. We
suggest that in protein folding an energetic barrier is essential to
separate the native from the unfolded conformations of a protein. This
barrier protects the ordered structure of a native protein against
continuous unfolding by diffusive chain motions and leads to apparent
two-state behavior.
Monomeric isomers of human interleukin 5 show that 1:1 receptor recruitment is sufficient for function
Authors: Li Jun , Cook Richard , Doyle Michael L. , Hensley Preston , McNulty Dean E. , Chaiken Irwin
Published: July 2002

The normally dimeric human interleukin 5 (IL-5) was re-engineered into two monomeric isomer forms to investigate mechanistic features of receptor recognition. One form, denoted GM1–IL-5, is a CD-loop expanded form, in which an 8-residue linker designed for flexibility was inserted between residues 85 and 86. The second, denoted DABC–IL-5, is a circularly permuted form of human IL-5 in which a chain discontinuity was introduced in the CD loop and the two consequent chain fragments were joined at the normal N and C termini by a di-glycyl linker. Both IL-5 isomers folded into stable monomers in solution as shown by sedimentation equilibrium and CD and formed an intrachain disulfide bond predicted from the structure of wild type IL-5. From titration microcalorimetry and optical biosensor analyses, both monomers were shown to interact with the IL-5 receptor α chain with 1:1 stoichiometry and affinities 30- to 40-fold weaker than for the dimeric wild type protein. And both monomers stimulated cell proliferation of human IL-5 receptor positive cells with a concentration dependence close to that of wild type. The data show that both monomeric and dimeric forms of IL-5 function through similar 1:1 receptor α chain recruitment processes and that it is the helical packing of the monomeric four-helix bundle unit in IL-5, rather than the helical connectivity itself, that appears to play the major role in presenting structural epitopes to trigger functional receptor activation.
A “knockdown” mutation created by cis-element gene targeting reveals the dependence of erythroid cell maturation on the level of transcription factor GATA-1
Authors: McDevitt Michael A. , Shivdasani Ramesh A. , Fujiwara Yuko , Yang Haidi , Orkin Stuart H.
Published: July 2002

The hematopoietic-restricted transcription factor GATA-1 is required for both mammalian erythroid cell and megakaryocyte differentiation. To define the mechanisms governing its transcriptional regulation, we replaced upstream sequences including a DNase I hypersensitive (HS) region with a neomycin-resistance cassette by homologous recombination in mouse embryonic stem cells and generated mice either harboring this mutation (neoΔHS) or lacking the selection cassette (ΔneoΔHS). Studies of the consequences of these targeted mutations provide novel insights into GATA-1 function in erythroid cells. First, the neoΔHS mutation leads to a marked impairment in the rate or efficiency of erythroid cell maturation due to a modest (4- to 5-fold) decrease in GATA-1 expression. Hence, erythroid differentiation is dose-dependent with respect to GATA-1. Second, since expression of GATA-1 from the ΔneoΔHS allele in erythroid cells is largely restored, transcription interference imposed by the introduced cassette must account for the “knockdown” effect of the mutation. Finally, despite the potency of the upstream sequences in conferring high-level, developmentally appropriate expression of transgenes in mice, other cis-regulatory elements within the GATA-1 compensate for its absence in erythroid cells. Our work illustrates the usefulness of targeted mutations to create knockdown mutations that may uncover important quantitative contributions of gene function not revealed by conventional knockouts.
Area-specific regulation of γ-aminobutyric acid type A receptor subtypes by thalamic afferents in developing rat neocortex
Authors: Paysan Jacques , Kossel Albrecht , Bolz Jürgen , Fritschy Jean-Marc
Published: July 2002

Targeting and innervation of the cerebral cortex by thalamic afferents is a key event in the specification of cortical areas. The molecular targets of thalamic regulation, however, have remained elusive. We now demonstrate that thalamic afferents regulate the expression of γ-aminobutyric acid type A (GABA
A
) receptors in developing rat neocortex, leading to the area-specific expression of receptor subtypes in the primary visual (V1) and somatosensory (S1) areas. Most strikingly, the α1- and α5-GABA
A
receptors exhibited a reciprocal expression pattern, which precisely reflected the distribution of thalamocortical afferents at postnatal day 7. Following unilateral lesions at the birth of the thalamic nuclei innervating V1 and S1 (lateral geniculate nucleus and ventrobasal complex, respectively), profound changes in subunit expression were detected 1 week later in the deprived cortical territories (layers III–IV of V1 and S1). The expression of the α1 subunit was strongly down-regulated in these layers to a level comparable to that in neighboring areas. Conversely, the α5 subunit was up-regulated and areal boundaries were no longer discernible in the lesioned hemisphere. Changes similar to the α5 subunit were also seen for the α2 and α3 subunits. These results indicate that the differential expression of GABA
A
receptor subtypes in developing neocortex is dependent on thalamic innervation, contributing to the emergence of functionally distinct areas.
Defective placental vasculogenesis causes embryonic lethality in VHL-deficient mice
Authors: Gnarra James R. , Ward Jerrold M. , Porter Forbes D. , Wagner Joseph R. , Devor Deborah E. , Grinberg Alex , Emmert-Buck Michael R. , Westphal Heiner , Klausner Richard D. , Linehan W. Marston
Published: July 2002

Inheritance of an inactivated form of the
VHL
tumor suppressor gene predisposes patients to develop von Hippel–Lindau disease, and somatic
VHL
inactivation is an early genetic event leading to the development of sporadic renal cell carcinoma. The
VHL
gene was disrupted by targeted homologous recombination in murine embryonic stem cells, and a mouse line containing an inactivated
VHL
allele was generated. While heterozygous
VHL
(+/−) mice appeared phenotypically normal,
VHL
−/− mice died
in utero
at 10.5 to 12.5 days of gestation (E10.5 to E12.5). Homozygous
VHL
−/− embryos appeared to develop normally until E9.5 to E10.5, when placental dysgenesis developed. Embryonic vasculogenesis of the placenta failed to occur in
VHL
−/− mice, and hemorrhagic lesions developed in the placenta. Subsequent hemorrhage in
VHL
−/− embryos caused necrosis and death. These results indicate that VHL expression is critical for normal extraembryonic vascular development.
Lipoxin A4stable analogs inhibit leukocyte rolling and adherence in the rat mesenteric microvasculature: Role of P-selectin
Authors: Scalia Rosario , Gefen Jonathan , Petasis Nicos A. , Serhan Charles N. , Lefer Allan M.
Published: July 2002

Three different stable lipoxin A4(LXA4) analogs (i.e., 16-phenoxy-LXA4-Me, 15-cyclohexyl-LXA4-Me, and 15-R/S-methyl-LXA4-Me) were studied for their ability to modulate leukocyte-endothelial cell interactions in the rat mesenteric microvasculature. Superfusion of the rat mesentery with 50 μmol/literNG-nitro-l-arginine methyl ester (l-NAME) caused a significant, time-dependent increase in leukocyte rolling (56 ± 8 cells/min;P< 0.01 vs. control) and leukocyte adherence (12.5 ± 1.2 cells/100 μm length of venule;P< 0.01 vs. control) after 120 min of superfusion. Concomitant superfusion of the rat mesentery with 10 nmol/liter of each of three lipoxin analogs consistently and markedly attenuatedl-NAME-induced leukocyte rolling to 10 ± 4 (P< 0.01), 4 ± 1 (P< 0.01), and 32 ± 7 (P< 0.05) cells/min, and adherence to 4 ± 0.8 (P< 0.01), 1.1 ± 0.4 (P< 0.01), and 7 ± 0.7 (P< 0.05) cells/100 μm length of venule (16-phenoxy-LXA4-Me, 15-cyclohexyl-LXA4-Me, and 15-R/S- methyl-LXA4-Me, respectively). No alterations of systemic blood pressure or mesenteric venular shear rates were observed in any group. Immunohistochemical up-regulation of P-selectin expression on intestinal venular endothelium was significantly increased (P< 0.01) after exposure tol-NAME, and this was significantly attenuated by these lipoxin analogs (P< 0.01). Thus,in vivosuperfusion of the rat mesentery with stable lipoxin analogs at 10 nmol/liter reducesl-NAME-induced leukocyte rolling and adherence in the mesenteric rat microvasculature by attenuating P-selectin expression. This anti-inflammatory mechanism may represent a novel and potent regulatory action of lipoxins on the immune system.
Effects of methionine on the cytoplasmic distribution of actin and tubulin during neural tube closure in rat embryos
Authors: Moephuli Shadrack R. , Klein Norman W. , Baldwin Michael T. , Krider Hallie M.
Published: July 2002

Research has previously shown that, without methionine supplements, neural tube proteins of rat embryos cultured on bovine sera were hypomethylated and neural tubes failed to close. In the present study, to identify the proteins that became methylated during neurulation, rat embryos were first cultured on methionine-deficient bovine serum for 40 hr, then incubated with puromycin for 1 hr, and, finally, incubated with [
methyl
-
14
C]methionine and puromycin for 5 hr. On the basis of molecular weights, isoelectric points, and Western immunoblots, the methyl-
14
C-labeled proteins were identified as actin, αβ-tubulin, and neurofilament L. Indirect immunofluorescence studies indicated that without the addition of methionine to the culture, localization of actin and αβ-tubulin in the basal cytoplasm did not occur and these neuroepithelial cells lost their columnar morphology.
Characterization of the CHD family of proteins
Authors: Woodage Trevor , Basrai Munira A. , Baxevanis Andreas D. , Hieter Philip , Collins Francis S.
Published: July 2002

The murine gene
CHD1
(
MmCHD1
) was previously isolated in a search for proteins that bound a DNA promoter element. The presence of chromo (chromatin organization modifier) domains and an SNF2-related helicase/ATPase domain led to speculation that this gene regulated chromatin structure or gene transcription. This study describes the cloning and characterization of three novel human genes related to
MmCHD1
. Examination of sequence databases produced several more related genes, most of which were not known to be similar to
MmCHD1
, yielding a total of 12 highly conserved CHD genes from organisms as diverse as yeast and mammals. The major region of sequence variation is in the C-terminal part of the protein, a region with DNA-binding activity in MmCHD1. Targeted deletion of
ScCHD1
, the sole
Saccharomyces cerevesiae
CHD gene, was performed with deletion strains being less sensitive than wild type to the cytotoxic effect of 6-azauracil. This finding suggested that enhanced transcriptional arrest at RNA polymerase II pause sites due to 6-azauracil-induced nucleotide pool depletion was reduced in the deletion strain and that
ScCHD1
inhibited transcription. This observation, along with the known roles of other proteins with chromo or SNF2-related helicase/ATPase domains, suggests that alteration of gene expression by CHD genes might occur by modifications of chromatin structure, with altered access of the transcriptional apparatus to its chromosomal DNA template.
Inhibition of HIV type 1 infectivity by constrained α-helical peptides: Implications for the viral fusion mechanism
Authors: Judice J. Kevin , Tom Jeffrey Y. K. , Huang Wei , Wrin Terri , Vennari Joann , Petropoulos Christos J. , McDowell Robert S.
Published: July 2002

Linear peptides derived from the membrane proximal region of the gp41 ectodomain are effective inhibitors of HIV type 1 (HIV-1)-mediated fusion events. These inhibitory peptides lack structure in solution, rendering mechanistic interpretation of their activity difficult. Using structurally constrained analogs of these molecules, we demonstrate that the peptides inhibit infectivity by adopting a helical conformation. Moreover, we show that a specific face of the helix must be exposed to block viral infectivity. Recent crystal structures show that the region of gp41 corresponding to the inhibitory peptides is helical and uses the analogous face to pack against a groove formed by an N-terminal coiled-coil trimer. Our results provide a direct link between the inhibition of HIV-1 infectivity by these peptides and the x-ray structures, and suggest that the conformation of gp41 observed by crystallography represents the fusogenic state. Other agents that block HIV-1 infectivity by binding to this groove may hold promise for the treatment of AIDS.
Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer
Authors: Pitard Bruno , Aguerre Olivier , Airiau Marc , Lachagès Anne-Marie , Boukhnikachvili Tsiala , Byk Gérardo , Dubertret Catherine , Herviou Christian , Scherman Daniel , Mayaux Jean-François , Crouzet Joël
Published: July 2002

Gene therapy is based on the vectorization of genes to target cells and their subsequent expression. Cationic amphiphile-mediated delivery of plasmid DNA is the nonviral gene transfer method most often used. We examined the supramolecular structure of lipopolyamine/plasmid DNA complexes under various condensing conditions. Plasmid DNA complexation with lipopolyamine micelles whose mean diameter was 5 nm revealed three domains, depending on the lipopolyamine/plasmid DNA ratio. These domains respectively corresponded to negatively, neutrally, and positively charged complexes. Transmission electron microscopy and x-ray scattering experiments on complexes originating from these three domains showed that although their morphology depends on the lipopolyamine/plasmid DNA ratio, their particle structure consists of ordered domains characterized by even spacing of 80 Å, irrespective of the lipid/DNA ratio. The most active lipopolyamine/DNA complexes for gene transfer were positively charged. They were characterized by fully condensed DNA inside spherical particles (diameter: 50 nm) sandwiched between lipid bilayers. These results show that supercoiled plasmid DNA is able to transform lipopolyamine micelles into a supramolecular organization characterized by ordered lamellar domains.
Catalysis of protein folding by symmetric chaperone complexes
Authors: Sparrer Helmut , Rutkat Kerstin , Buchner Johannes
Published: July 2002

The GroE chaperones of
Escherichia coli
assist protein folding under physiological and heat shock conditions in an ATP-dependent way. Although a number of details of assisted folding have been elucidated, the molecular mechanism of the GroE cycle remains unresolved. Here we present an experimental system that allows the direct analysis of the GroE-mediated folding cycle under stringent conditions. We demonstrate that the GroE proteins efficiently catalyze the folding of kinetically trapped folding intermediates of a mutant of maltose-binding protein (MBP Y283D) in an ATP-dependent way. GroES plays a key role in this reaction cycle, accelerating the folding of the substrate protein MBP Y283D up to 50-fold. Interestingly, catalysis of the folding reaction requires the formation of symmetrical football-shaped GroEL·GroES
2
particles and the intermediate release of the nonnative protein from the chaperone complex. Our results show that, in the presence of GroES, the complex architecture of the GroEL toroids allows maintenance of two highly interregulated rings simultaneously active in protein folding.
Directin vivomeasurement of targeted binding in a human tumor xenograft
Authors: Berk David A. , Yuan Fan , Leunig Michael , Jain Rakesh K.
Published: July 2002

Binding is crucial to the function of most biologically active molecules, but difficult to quantify directly in living tissue. To this end, fluorescence recovery after photobleaching was used to detect the immobilization of fluorescently labeled ligand caused by binding to receptorsin vivo. Measurements of mAb affinity to target antigen within human tumor xenografts revealed a saturable binding isotherm, from which anin vivocarcinoembryonic antigen density of 0.56 nmol/g (5.0 × 105/cell) and an association constant ofKa⩽ 4 × 107M−1were estimated. The present method can be adapted forin vivostudies of cell signaling, targeted drugs, gene therapy, and other processes involving receptor-ligand binding.
Ras-dependent pathways induce obstructive hypertrophy in echo-selected transgenic mice
Authors: Gottshall Kim R. , Hunter John J. , Tanaka Nobuaki , Dalton Nancy , Becker K. David , Ross John , Chien Kenneth R.
Published: July 2002

To overcome the genetic and interindividual variability frequently noted in complex phenotypes, we used echocardiographic selection to develop a substrain of myosin light chain (MLC)–Ras (RAS) transgenic mice with an enhanced ventricular hypertrophic phenotype. These echo-selected mice were then compared with wild-type (WT) animals and a pressure overload hypertrophy model (transverse aortic constriction; TAC). Echocardiography demonstrated increased wall thickness in RAS compared with the other groups. We developed novel miniaturized physiological technology to quantitatively identify
in vivo
intraventricular gradients; increased systolic Doppler velocity was seen in the left ventricle (LV) in 69% of RAS vs. none of WT or TAC. Intracavitary pressure gradients were present in 3 of 10 RAS vs. none of TAC or WT. Passive diastolic LV stiffness was not different among the three groups. Myofibrillar disarray was present in all RAS animals and was significantly more extensive (21.7% area fraction) than in TAC (1.5%) or WT (0.0%). RAS mice had selective induction of natriuretic peptide genes in the LV, a pattern distinct from that induced by pressure overload. Juvenile mortality was significantly increased in the offspring of echo-selected RAS parents. We conclude that adaptation of echocardiography to the mouse permits selection for cardiac phenotypes, and that selectively inbred MLC–Ras transgenic mice faithfully reproduce the molecular, physiological, and pathological features of human hypertrophic cardiomyopathy (HCM). Because previous studies support the concept that hypertrophy in human HCM is secondary to dysfunction created by sarcomeric protein mutations, the current studies suggest that Ras-dependent pathways might play a similar role in forms of human HCM.
DNA methylation and the promotion of flowering by vernalization
Authors: Finnegan E. J. , Genger R. K. , Kovac K. , Peacock W. J. , Dennis E. S.
Published: July 2002

We have tested the hypothesis that the promotion of flowering by prolonged exposure to low temperatures (vernalization) is mediated by DNA demethylation [Burn, J. E., Bagnall, D. J., Metzger, J. M., Dennis, E. S. & Peacock, W. J. (1993)
Proc. Natl. Acad. Sci. USA
90, 287–291]. Arabidopsis plants that have reduced levels of DNA methylation because of the presence of a methyltransferase (
METI
) antisense gene flowered earlier than untransformed control plants, without the need for a cold treatment. Decreased DNA methylation mutants (
ddm1
) also flowered earlier than the wild-type progenitor under conditions where they respond to vernalization. We conclude that demethylation of DNA is sufficient to cause early flowering, and we have found that the promotion of flowering is directly proportional to the decrease in methylation in
METI
antisense lines. The early-flowering phenotype was inherited in sexual progeny, even when the antisense transgene had been lost by segregation. Methyltransferase antisense plants with low DNA methylation levels responded to a low-temperature treatment by flowering even earlier than their untreated siblings indicating that the promotion of flowering by cold and by demethylation was additive when neither treatment saturated the early-flowering response. As in untransformed control plants, the cold-induced early-flowering signal was reset in progeny of
METI
antisense plants. These observations suggest that the demethylation brought about by a
METI
antisense can account for some properties of vernalization, but not for the need for revernalization in each generation.
Intramolecular vibrational dephasing obeys a power law at intermediate times
Authors: Gruebele M.
Published: July 2002

Experimental intramolecular vibrational dephasing transients for several large organic molecules are reanalyzed. Fits to the experimental data, as well as full numerical quantum calculations with a factorized potential surface for all active degrees of freedom of fluorene indicate that power law decays, not exponentials, occur at intermediate times. The results support a proposal that power law decays describe vibrational dephasing dynamics in large molecules at intermediate times because of the local nature of energy flow.
Reexamination of the mechanism of hydroxyl radical adducts formed from the reaction between familial amyotrophic lateral sclerosis-associated Cu,Zn superoxide dismutase mutants and H
2
O
2
Authors: Singh Ravinder J. , Karoui Hakim , Gunther Michael R. , Beckman Joseph S. , Mason Ronald P. , Kalyanaraman B.
Published: July 2002

Amyotrophic lateral sclerosis (ALS) involves the progressive
degeneration of motor neurons in the spinal cord and motor cortex.
Mutations to Cu,Zn superoxide dismutase (SOD) linked with familial ALS
are reported to increase hydroxyl radical adduct formation from
hydrogen peroxide as measured by spin trapping with
5,5′-dimethyl-1-pyrrolline
N
-oxide (DMPO). In the
present study, we have used oxygen-17-enriched water and
H
2
O
2
to reinvestigate the mechanism of
DMPO/
⋅
OH formation from the SOD and SOD mutants. The relative
ratios of DMPO/
⋅
17
OH and
DMPO/
⋅
16
OH formed in the Fenton reaction were 90%
and 10%, respectively, reflecting the ratios of
H
2
17
O
2
to
H
2
16
O
2
. The reaction of the WT SOD
with H
2
17
O
2
in
bicarbonate/CO
2
buffer yielded 63%
DMPO/
⋅
17
OH and 37%
DMPO/
⋅
16
OH. Similar results were obtained from the
reaction between familial ALS SOD mutants and
H
2
17
O
2
:
DMPO/
⋅
17
OH (64%); DMPO/
⋅
16
OH
(36%) from A4V and DMPO/
⋅
17
OH (62%); and
DMPO/
⋅
16
OH (38%) from G93A. These results were
confirmed further by using 5-diethoxyphosphoryl-5-methyl-1-pyrroline
N
-oxide spin trap, a phosphorylated analog of DMPO.
Contrary to earlier reports, the present results indicate that a
significant fraction of DMPO/
⋅
OH formed during the reaction of
SOD and familial ALS SOD mutants with H
2
O
2
is
derived from the incorporation of oxygen from water due to oxidation of
DMPO to DMPO/
⋅
OH presumably via DMPO radical cation. No
differences were detected between WT and mutant SODs, neither in the
concentration of DMPO/
⋅
OH or DEPMPO/
⋅
OH formed nor in
the relative incorporation of oxygen from H
2
O
2
or water.
Conformational changes couple Na
+
and glucose transport
Authors: Loo Donald D. F. , Hirayama Bruce A. , Gallardo Elsa M. , Lam Jason T. , Turk Eric , Wright Ernest M.
Published: July 2002

The mechanism by which cotransport proteins couple their substrates across cell membranes is not known. A commonly proposed model is that cotransport results from ligand-induced conformational transitions that change the accessibility of ligand-binding sites from one side of the membrane to the other. To test this model, we have measured the accessibility of covalent probes to a cysteine residue (Q457C) placed in the putative sugar-translocation domain of the Na
+
/glucose cotransporter (SGLT1). The mutant protein Q457C was able to transport sugar, but transport was abolished after alkylation by methanethiosulfonate reagents. Alkylation blocked sugar translocation but not sugar binding. Accessibility of Q457C to alkylating reagents required external Na
+
and was blocked by external sugar and phlorizin. The voltage dependence of accessibility was directly correlated with the presteady–state charge movement of SGLT1. Voltage-jump experiments with rhodamine-6-maleimide-labeled Q457C showed that the time course and level of changes in fluorescence closely followed the presteady–state charge movement. We conclude that conformational changes are responsible for the coupling of Na
+
and sugar transport and that Q457 plays a critical role in sugar translocation by SGLT1.
Myosin conformational states determined by single fluorophore polarization
Authors: Warshaw David M. , Hayes Eric , Gaffney Donald , Lauzon Anne-Marie , Wu Junru , Kennedy Guy , Trybus Kathleen , Lowey Susan , Berger Christopher
Published: July 2002

Muscle contraction is powered by the interaction of the molecular motor myosin with actin. With new techniques for single molecule manipulation and fluorescence detection, it is now possible to correlate, within the same molecule and in real time, conformational states and mechanical function of myosin. A spot-confocal microscope, capable of detecting single fluorophore polarization, was developed to measure orientational states in the smooth muscle myosin light chain domain during the process of motion generation. Fluorescently labeled turkey gizzard smooth muscle myosin was prepared by removal of endogenous regulatory light chain and re-addition of the light chain labeled at cysteine-108 with the 6-isomer of iodoacetamidotetramethylrhodamine (6-IATR). Single myosin molecule fluorescence polarization data, obtained in a motility assay, provide direct evidence that the myosin light chain domain adopts at least two orientational states during the cyclic interaction of myosin with actin, a randomly disordered state, most likely associated with myosin whereas weakly bound to actin, and an ordered state in which the light chain domain adopts a finite angular orientation whereas strongly bound after the powerstroke.
Synergy between simulation and experiment in describing the energy landscape of protein folding
Authors: Ladurner Andreas G. , Itzhaki Laura S. , Daggett Valerie , Fersht Alan R.
Published: July 2002

Experimental data from protein engineering studies and NMR spectroscopy have been used by theoreticians to develop algorithms for helix propensity and to benchmark computer simulations of folding pathways and energy landscapes. Molecular dynamic simulations of the unfolding of chymotrypsin inhibitor 2 (CI2) have provided detailed structural models of the transition state ensemble for unfolding/folding of the protein. We now have used the simulated transition state structures to design faster folding mutants of CI2. The models pinpoint a number of unfavorable local interactions at the carboxyl terminus of the single α-helix and in the protease-binding loop region of CI2. By removing these interactions or replacing them with stabilizing ones, we have increased the rate of folding of the protein up to 40-fold (τ = 0.4 ms). This correspondence, and other examples of agreement between experiment and theory in general, Φ-values and molecular dynamics simulations, in particular, suggest that significant progress has been made toward describing complete folding pathways at atomic resolution by combining experiment and simulation.
A high throughput screen to identify secreted and transmembrane proteins involved in
Drosophila
embryogenesis
Authors: Kopczynski Casey C. , Noordermeer Jasprina N. , Serano Thomas L. , Chen Wei-Yu , Pendleton John D. , Lewis Suzanna , Goodman Corey S. , Rubin Gerald M.
Published: July 2002

Secreted and transmembrane proteins play an essential role in intercellular communication during the development of multicellular organisms. Because only a small number of these genes have been characterized, we developed a screen for genes encoding extracellular proteins that are differentially expressed during
Drosophila
embryogenesis. Our approach utilizes a new method for screening large numbers of cDNAs by whole-embryo
in situ
hybridization. The cDNA library for the screen was prepared from rough endoplasmic reticulum-bound mRNA and is therefore enriched in clones encoding membrane and secreted proteins. To increase the prevalence of rare cDNAs in the library, the library was normalized using a method based on cDNA hybridization to genomic DNA-coated beads. In total, 2,518 individual cDNAs from the normalized library were screened by
in situ
hybridization, and 917 of these cDNAs represent genes differentially expressed during embryonic development. Sequence analysis of 1,001 cDNAs indicated that 811 represent genes not previously described in
Drosophila
. Expression pattern photographs and partial DNA sequences have been assembled in a database publicly available at the Berkeley
Drosophila
Genome Project website (
http://fruitfly.berkeley.edu
). The identification of a large number of genes encoding proteins involved in cell–cell contact and signaling will advance our knowledge of the mechanisms by which multicellular organisms and their specialized organs develop.
Equilibrium distributions of microsatellite repeat length resulting from a balance between slippage events and point mutations
Authors: Kruglyak Semyon , Durrett Richard T. , Schug Malcolm D. , Aquadro Charles F.
Published: July 2002

We describe and test a Markov chain model of microsatellite evolution that can explain the different distributions of microsatellite lengths across different organisms and repeat motifs. Two key features of this model are the dependence of mutation rates on microsatellite length and a mutation process that includes both strand slippage and point mutation events. We compute the stationary distribution of allele lengths under this model and use it to fit DNA data for di-, tri-, and tetranucleotide repeats in humans, mice, fruit flies, and yeast. The best fit results lead to slippage rate estimates that are highest in mice, followed by humans, then yeast, and then fruit flies. Within each organism, the estimates are highest in di-, then tri-, and then tetranucleotide repeats. Our estimates are consistent with experimentally determined mutation rates from other studies. The results suggest that the different length distributions among organisms and repeat motifs can be explained by a simple difference in slippage rates and that selective constraints on length need not be imposed.
One face of a transmembrane helix is crucial in mechanosensitive channel gating
Authors: Ou Xiaorong , Blount Paul , Hoffman Robert J. , Kung Ching
Published: July 2002

MscL is a mechanosensitive channel in bacteria that responds directly to membrane tension by opening a large conductance pore. To determine functionally important residues within this molecule, we have randomly mutagenized
mscL
, expressed the genes in living bacteria, and screened for gain-of-function mutants with hampered growth. Expression of these genes caused leakage of cytoplasmic solutes on little or no hypo-osmotic stress. In excised patches, the mutant channels gated at membrane tensions that are less than that required for the gating of the wild-type MscL. Hence, the data suggest that the slowed or no-growth phenotype is caused by solute loss because of inappropriate gating of the channel. Most of the mutations mapped to the first transmembrane domain. When this domain is modeled as an α-helix, the most severe mutations are substitutions of smaller amino acids (three glycines and one valine) on one facet, suggesting an important role for this structure in MS channel gating.
The kinetics and shear threshold of transient and rolling interactions of L-selectin with its ligand on leukocytes
Authors: Alon Ronen , Chen Shuqi , Fuhlbrigge Robert , Puri Kamal D. , Springer Timothy A.
Published: July 2002

The kinetics of rolling and transient adhesions through selectins may depend on the kinetics and mechanical properties of the selectin:ligand bond, as well as on cellular properties including receptor-anchoring to the cell membrane and cytoskeleton. Kinetics are known to depend on the selectin and may also be ligand dependent. Here, we study the kinetics of transient and rolling interactions of leukocytes with L-selectin immobilized on a substrate. Remarkably, all properties examined are similar to those seen when the sidedness is opposite, i.e., when the L-selectin ligand is on the substrate and when the ligand is isolated from HEV rather than present on leukocytes. The similar properties include rolling velocity, a threshold shear stress above 0.4 dyn/cm2required to support rolling, ak°offof 7.0 to 6.8 s−1for the L-selectin tether bond, and a mechanical bond length of 0.24 to 0.20 Å. Our results argue against a model in which L-selectin shedding mediates rolling. Furthermore, the fast and force-resistant kinetic properties suggest that L-selectin is specialized dynamically for tethering leukocytes to vessel walls and adherent leukocytes.
Conformational gating of the electron transfer reaction Q
A
−
⋅
Q
B
→ Q
A
Q
B
−
⋅
in bacterial reaction centers of
Rhodobacter sphaeroides
determined by a driving force assay
Authors: Graige M. S. , Feher G. , Okamura M. Y.
Published: July 2002

The mechanism of the electron transfer reaction, Q
A
−
⋅
Q
B
→ Q
A
Q
B
−
⋅
,
was studied in isolated reaction centers from the photosynthetic bacterium
Rhodobacter sphaeroides
by replacing the native Q
10
in the Q
A
binding site with quinones having different redox potentials. These substitutions are expected to change the intrinsic electron transfer rate by changing the redox free energy (i.e., driving force) for electron transfer without affecting other events that may be associated with the electron transfer (e.g., protein dynamics or protonation). The electron transfer from Q
A
−
⋅
to Q
B
was measured by three independent methods: a functional assay involving cytochrome c
2
to measure the rate of Q
A
−
⋅
oxidation, optical kinetic spectroscopy to measure changes in semiquinone absorption, and kinetic near-IR spectroscopy to measure electrochromic shifts that occur in response to electron transfer. The results show that the rate of the observed electron transfer from Q
A
−
⋅
to Q
B
does not change as the redox free energy for electron transfer is varied over a range of 150 meV. The strong temperature dependence of the observed rate rules out the possibility that the reaction is activationless. We conclude, therefore, that the independence of the observed rate on the driving force for electron transfer is due to conformational gating, that is, the rate limiting step is a conformational change required before electron transfer. This change is proposed to be the movement, controlled kinetically either by protein dynamics or intermolecular interactions, of Q
B
by ≈5 Å as observed in the x-ray studies of Stowell
et al.
[Stowell, M. H. B., McPhillips, T. M., Rees, D. C., Soltis, S. M., Abresch, E. & Feher, G. (1997)
Science
276, 812–816].
Cleavage motifs of the yeast 20S proteasome β subunits deduced from digests of enolase 1
Authors: Nussbaum Alexander K. , Dick Tobias P. , Keilholz Wieland , Schirle Markus , Stevanović Stefan , Dietz Klaus , Heinemeyer Wolfgang , Groll Michael , Wolf Dieter H. , Huber Robert , Rammensee Hans-Georg , Schild Hansjörg
Published: July 2002

The 436-amino acid protein enolase 1 from yeast was degradedin vitroby purified wild-type and mutant yeast 20S proteasome particles. Analysis of the cleavage products at different times revealed a processive degradation mechanism and a length distribution of fragments ranging from 3 to 25 amino acids with an average length of 7 to 8 amino acids. Surprisingly, the average fragment length was very similar between wild-type and mutant 20S proteasomes with reduced numbers of active sites. This implies that the fragment length is not influenced by the distance between the active sites, as previously postulated. A detailed analysis of the cleavages also allowed the identification of certain amino acid characteristics in positions flanking the cleavage site that guide the selection of the P1 residues by the three active β subunits. Because yeast and mammalian proteasomes are highly homologous, similar cleavage motifs might be used by mammalian proteasomes. Therefore, our data provide a basis for predicting proteasomal degradation products from which peptides are sampled by major histocompatibility complex class I molecules for presentation to cytotoxic T cells.
Involvement of focal adhesion kinase in invasin-mediated uptake
Authors: Alrutz Michael A. , Isberg Ralph R.
Published: July 2002

High-efficiency entry of the enteropathogenic bacterium
Yersinia pseudotuberculosis
into nonphagocytic cells is mediated by the bacterial outer membrane protein invasin. Invasin-mediated uptake requires high affinity binding of invasin to multiple β1 chain integrin receptors on the host eukaryotic cell. Previous studies using inhibitors have indicated that high-efficiency uptake requires tyrosine kinase activity. In this paper we demonstrate a requirement for focal adhesion kinase (FAK) for invasin-mediated uptake. Overexpression of a dominant interfering form of FAK reduced the amount of bacterial entry. Specifically, the autophosphorylation site of FAK, which is a reported site of c-Src kinase binding, is required for bacterial internalization, as overexpression of a derivative lacking the autophosphorylation site had a dominant interfering effect as well. Cultured cells expressing interfering variants of Src kinase also showed reduced bacterial uptake, demonstrating the involvement of a Src-family kinase in invasin-promoted uptake.
Complexities in the genetic structure of
Anopheles gambiae
populations in west Africa as revealed by microsatellite DNA analysis
Authors: Lanzaro Gregory C. , Touré Yeya T. , Carnahan John , Zheng Liangbiao , Dolo Guimogo , Traoré Sekou , Petrarca Vincenzo , Vernick Kenneth D. , Taylor Charles E.
Published: July 2002

Chromosomal forms of
Anopheles gambiae
, given the informal designations Bamako, Mopti, and Savannah, have been recognized by the presence or absence of four paracentric inversions on chromosome
2
. Studies of karyotype frequencies at sites where the forms occur in sympatry have led to the suggestion that these forms represent species. We conducted a study of the genetic structure of populations of
An
.
gambiae
from two villages in Mali, west Africa. Populations at each site were composed of the Bamako and Mopti forms and the sibling species,
Anopheles arabiensis
. Karyotypes were determined for each individual mosquito and genotypes at 21 microsatellite loci determined. A number of the microsatellites have been physically mapped to polytene chromosomes, making it possible to select loci based on their position relative to the inversions used to define forms. We found that the chromosomal forms differ at all loci on chromosome
2
, but there were few differences for loci on other chromosomes. Geographic variation was small. Gene flow appears to vary among different regions within the genome, being lowest on chromosome
2
, probably due to hitchhiking with the inversions. We conclude that the majority of observed genetic divergence between chromosomal forms can be explained by forces that need not involve reproductive isolation, although reproductive isolation is not ruled out. We found low levels of gene flow between the sibling species
Anopheles gambiae
and
Anopheles arabiensis
, similar to estimates based on observed frequencies of hybrid karyotypes in natural populations.
Identification of
morc
(
microrchidia
), a mutation that results in arrest of spermatogenesis at an early meiotic stage in the mouse
Authors: Watson Mark L. , Zinn Andrew R. , Inoue Norimitsu , Hess Karl D. , Cobb John , Handel Mary Ann , Halaban Ruth , Duchene Clark C. , Albright George M. , Moreadith Randall W.
Published: July 2002

The
microrchidia
, or
morc
, autosomal recessive mutation results in the arrest of spermatogenesis early in prophase I of meiosis. The
morc
mutation arose spontaneously during the development of a mouse strain transgenic for a tyrosinase cDNA construct.
Morc
−/− males are infertile and have grossly reduced testicular mass, whereas −/− females are normal, indicating that the
Morc
gene acts specifically during male gametogenesis. Immunofluorescence to synaptonemal complex antigens demonstrated that −/− male germ cells enter meiosis but fail to progress beyond zygotene or leptotene stage. An apoptosis assay revealed massive numbers of cells undergoing apoptosis in testes of −/− mice. No other abnormal phenotype was observed in mutant animals, with the exception of eye pigmentation caused by transgene expression in the retina. Spermatogenesis is normal in +/− males, despite significant transgene expression in germ cells. Genomic analysis of −/− animals indicates the presence of a deletion adjacent to the transgene. Identification of the gene inactivated by the transgene insertion may define a novel biochemical pathway involved in mammalian germ cell development and meiosis.
Plasticity of the neoplastic phenotype
in vivo
is regulated by epigenetic factors
Authors: McCullough Karen D. , Coleman William B. , Ricketts Sharon L. , Wilson Julie W. , Smith Gary J. , Grisham Joe W.
Published: July 2002

Age of host and transplantation-site microenvironment influence the tumorigenic potential of neoplastically transformed liver epithelial cells. Tumorigenic BAG2-GN6TF rat liver epithelial cells consistently form tumors at ectopic sites, but differentially express tumorigenicity or hepatocytic differentiation in the liver depending on host age and route of cell transplantation into the liver. Direct inoculation into host livers concentrates tumor cells locally, resulting in undifferentiated tumors near the transplantation site in both young (3-month-old) and old (18-month-old) rats. Transplantation-site tumors regress within 1 month in the livers of young rats, but grow progressively in old rats. However, inoculation of cells into the spleen distributes transplanted cells individually throughout the liver, resulting in hepatocytic differentiation by tumor cells with concomitant suppression of their tumorigenicity in young rats. When transplanted into livers of old rats by splenic inoculation, or when young hepatic-transplant recipients are allowed to age, hepatocytic progeny of BAG2-GN6TF cells proliferate to form foci, suggesting that the liver microenvironment of old rats incompletely regulates the proliferation and differentiation of tumor cell-derived hepatocytes. Upon removal from the liver, BAG2-GN6TF-derived hepatocytes revert to an undifferentiated, aggressively tumorigenic phenotype. We posit that the spectrum between normal differentiation and malignant potential of these cells reflects the dynamic interaction of the specific transformation-related genotype of the cells and the characteristics of the tissue microenvironment at the transplantation site. Changes in the tissue milieu, such as those that accompany normal aging, may determine the ability of a genetically aberrant cell to produce a tumor.
The JC and BK human polyoma viruses appear to be recent introductions to some South American Indian tribes: There is no serological evidence of cross-reactivity with the simian polyoma virus SV40
Authors: Major Eugene O. , Neel James V.
Published: July 2002

In an effort to understand the unusual cytogenetic damage earlier encountered in the Yanomama Indians, plasma samples from 425 Amerindians representing 14 tribes have been tested for hemagglutination inhibition antibodies to the human JC polyoma virus and from 369 Amerinds from 13 tribes for hemagglutination inhibition antibodies to the human BK polyoma virus. There is for both viruses highly significant heterogeneity between tribes for the prevalence of serum antibody titers ≥1/40, the pattern of infection suggesting that these two viruses only relatively recently have been introduced into some of these tribes. Some of these samples, from populations with no known exposure to the simian polyoma virus SV40, also were tested for antibodies to this virus by using an immunospot assay. In contrast to the findings of Brown
et al.
(Brown, P., Tsai, T. & Gajdusek, D. C. (1975)
Am. J. Epidemiol.
102, 331–340), none of the samples was found to possess antibodies to SV40. In addition, no significant titers to SV40 were found in a sample of 97 Japanese adults, many of whom had been found to exhibit elevated titers to the JC and BK viruses. This study thus suggests that these human sera contain significant antibody titers to the human polyoma viruses JC and BK but do not appear to contain either cross-reactive antibodies to SV40 or primary antibodies resulting from SV40 infection.
The rat gene homologous to the human gene 9–27 is involved in the development of the mammary gland
Authors: Zucchi I. , Montagna C. , Susani L. , Vezzoni P. , Dulbecco R.
Published: July 2002

We have developed a model system for studying differentiation in the mammary gland, by using two clonal cultures deriving from a rat breast adenocarcinoma. They differ in the ability to form domes, structures the significance of which is unknown. By using the subtractive cDNA library approach, we isolated a cDNA that is highly expressed in the dome-forming cells, and identical to the rat8 gene and highly homologous to the human 9–27 gene. Antisense treatment of the dome-forming cells specifically and reproducibly abolishes dome formation, while forced expression of the gene in non-dome-forming cells causes morphological changes suggestive of “flat” domes.In situhybridization on rat tissues shows that the gene is expressed in epithelia, especially in those forming tubular structures, suggesting a relatedness between these structures and domes. Cytokeratin 8 and E cadherin are strongly expressed in the domes but not outside them, suggesting that the rat8 gene triggers the cells to express molecules that tighten the lateral connections between the cells; the process is likely to parallel that occurring during the differentiation of the mammary gland.